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Research

Pipe Flowrate Measurement: Problem Solving

A spray tank system is engineered to uniformly distribute a pest-control liquid across plants by using a pressurized mechanism. The tank, pressurized to 150 kPa, holds the pesticide at a height of 0.80 meters. Liquid flows from the tank through a 1.9 meter pipe with a diameter of 0.015 meters, angled at 0.698 radians, ultimately reaching a 0.007 meter nozzle that sprays the pesticide. Accurate calculation of the system&#39;s flow rate is crucial to ensure uniform application, and this is achieved by first determining the flow velocity within the connecting pipe.
The design requires an understanding of fluid mechanics principles. At the outset, the velocity of the fluid in the tank at Point 1 is zero, with the pressure set to 150 kPa. Point 2, located at the nozzle, is assumed to be open to the atmosphere, with a pressure of zero. The height difference between Points 1 and 2, calculated using the 0.698-radian pipe angle and pipe length, determines the elevation change component in the Bernoulli equation. This height difference is found by multiplying the pipe length by the sine of the inclination angle.
To connect the parameters at Points 1 and 2, a modified Bernoulli equation is used, accounting for energy losses due to friction and the nozzle&#39;s loss coefficient. The equation expresses the pressure, velocity, and elevation terms for both points as follows:
<img alt="Equation 1" src="/files/ftp_upload/18085/18085_PT_equation_1.svg" style="text-align: center; height: 30px;" />
Here, p represents pressure, &#961; the fluid density, V the velocity, g the gravitational acceleration, z1 the height of the pesticide inside the tank, and z2&#160; the nozzle height above the bottom of the tank. With values substituted for these terms, the flow velocity in the pipe at Point 1 is solved.
After calculating the pipe&#39;s flow velocity, the flow rate, Q, is determined by multiplying this velocity by the cross-sectional area A of the pipe:
<img alt="Equation 2" src="/files/ftp_upload/18085/18085_PT_equation_2.svg" style="text-align: center; height: 11px;" />
Where A is:
<img alt="Equation 3" src="/files/ftp_upload/18085/18085_PT_equation_3.svg" style="text-align: center; height: 25px;" />
This flow rate calculation ensures that the pesticide is delivered at a consistent and effective rate, optimizing pest control application.

Consider a spray tank system designed to evenly apply pest-control liquid to plants.
The system includes a pressurized tank at 150 kPa filled with ...

Pipe Flowrate Measurement

In pipe flow measurement, orifice, nozzle, and Venturi meters are commonly used to determine fluid flowrates by constricting the flow area, which increases fluid velocity and reduces pressure. This pressure difference, governed by Bernoulli&#39;s principle and adjusted for real-world conditions, is essential for calculating flowrate. Each meter type is suited to specific applications based on accuracy, efficiency, and compatibility with various flow conditions.
The orifice meter is a simple, cost-effective device consisting of a flat plate with a central hole that creates a measurable pressure drop. This device is popular in applications like water treatment, oil and gas processing, and general industrial use where moderate accuracy is acceptable. However, due to head loss from turbulent flow and area contraction, a discharge coefficient is used to correct the idealized flow equation. The flowrate through an orifice meter is proportional to the square root of the pressure difference, accounting for fluid density, orifice area, and the ratio of the hole diameter to the pipe diameter.
The nozzle meter improves on the orifice meter by using a contoured design that reduces flow separation and turbulence, resulting in a smoother flow and increased measurement accuracy. It is often used for high-velocity flows, such as in steam or gas pipelines, chemical processing, and power plants. With a higher discharge coefficient than the orifice meter, the nozzle meter provides improved precision with less energy loss.
The Venturi meter offers the highest accuracy among the three types. It is ideal for applications requiring minimal head loss and reliable measurements, such as water supply systems, natural gas pipelines, and wastewater management. Its smooth contraction and gradual expansion reduce flow separation, conserving energy and enabling accurate flowrate calculations even in low-pressure systems. The discharge coefficient for a Venturi meter, which depends on factors like the ratio of the throat to pipe diameter and flow characteristics, supports exact flow measurement but requires a more complex installation and is generally more costly.
Each meter type offers unique benefits, from essential flow monitoring to precise flow control, chosen based on the application&#39;s accuracy needs and specific conditions.

Flow measurement devices&#8212;such as orifice, nozzle, and Venturimeters&#8212;operate by narrowing the flow area. This increases fluid velocity and ...

Design Example: Designing a Residential Plumbing System

The design of residential plumbing systems requires carefully evaluating water demand, flow rates, and pressure dynamics to ensure both efficiency and reliability. The nature of water flow within pipes is defined by its Reynolds number, which classifies flow as either laminar (smooth) or turbulent.
<img alt="Equation 1" src="/files/ftp_upload/18086/18086_Equation_1.svg" style="text-align: center; height: 42px;" />
This dimensionless value depends on factors such as water velocity and pipe. Residential plumbing systems are designed to meet specific water demand and flow rate requirements based on occupancy and fixture needs.
Determining the nature of water flow &#8212; laminar or turbulent &#8212; requires calculating the Reynolds number, which depends on variables like pipe diameter and water velocity. In residential settings, flow is often turbulent, leading to higher energy loss due to friction, which impacts system efficiency. Energy losses, or head losses, arise as water moves through pipes. These losses are classified as either major or minor. Major losses from friction along pipe walls are calculated using the Darcy-Weisbach equation.
<img alt="Equation 2" src="/files/ftp_upload/18086/18086_Equation_2.svg" style="text-align: center; height: 42px;" />
The Darcy-Weisbach equation requires a friction factor, which is obtained from the Moody chart. This chart accounts for pipe roughness and flow characteristics, which significantly affect turbulent flow friction levels. Minor losses, in contrast, are caused by fittings, bends, and valves, each of which contributes resistance to flow. Each fitting is assigned a loss coefficient that quantifies its resistance and is factored into the total head loss.
<img alt="Equation 3" src="/files/ftp_upload/18086/18086_Equation_3.svg" style="text-align: center; height: 48px;" />
By adding major and minor losses, the overall head loss can be determined and managed so as to maintain adequate pressure, avoiding potential leaks or damage. Flow measurement devices like Venturi or orifice meters ensure the plumbing system provides the necessary flow rates. These meters measure flow rates by detecting pressure differences across constrictions in the pipe, confirming that water flow meets design specifications and household needs.

The residential plumbing system is designed by assessing the building&#39;s water demand and flow rate needs based on occupancy and fixture requirements.
...

Single Pipe Systems

In pipe flow analysis, problems are typically categorized into three types &#8212; Type I, Type II, and Type III &#8212; based on the known parameters and the desired outcome. Each type of problem addresses specific engineering requirements using fluid properties, pipe characteristics, and operational conditions.
In a Type I problem, fluid properties (density and viscosity), pipe characteristics (including diameter, length, and surface roughness), and the flow rate or average velocity are known. The objective is calculating the pressure drop or head loss needed to maintain the specified flow rate. For instance, in a heating system circulating hot water at 0.32 liters per second, knowing the system&#39;s fluid and pipe specifications allows engineers to determine the required pressure to achieve steady flow. Type I problems are common in designing HVAC systems, where maintaining consistent flow is essential for effective temperature control.
A Type II problem provides the applied pressure, fluid properties, and pipe details, to calculate the resulting flow rate. This type of analysis is used in applications such as garden irrigation, where engineers need to determine the water flow rate in a sprinkler system operating at a specified pressure, such as 276 kilopascals. Knowing the pipe dimensions and roughness, the flow rate reaching each sprinkler can be calculated to ensure adequate water distribution.
In a Type III problem, the known parameters include the pressure drop and desired flow rate, to determine the optimal pipe diameter to support efficient flow. This scenario is typical in cooling systems or industrial piping design, where engineers must size the pipe to achieve a flow rate of 0.19 liters per second, balancing efficiency with minimal energy loss. Choosing an appropriate pipe diameter in Type III problems ensures the required flow rate while minimizing pressure losses and pump energy demands.
By systematically categorizing pipe flow problems, engineers can streamline analysis and ensure optimal design and performance for fluid transport systems in heating, irrigation, and industrial applications.

In pipe flow problems, scenarios are grouped by known information and desired calculation and are typically classified as Type I, II, or III.
In a Type I ...

Minor Losses in Pipes

In pipe systems, minor losses refer to energy losses arising from components such as valves, bends, fittings, expansions, and other features that disrupt the steady flow of fluid. These disturbances cause energy dissipation through turbulence and resistance, which engineers quantify to manage system efficiency effectively.
Valves play a significant role in generating minor losses by obstructing or redirecting the fluid flow. When a valve is closed or partially closed, it restricts the flow path, creating additional resistance and inducing turbulence. The resulting energy dissipation depends on the valve&#39;s geometry, position, and flow path design. To quantify this effect, engineers use a loss coefficient, K, which scales with the square of the fluid velocity V as follows:
<img alt="Equation 1" src="/files/ftp_upload/18081/18081_Equation_1.svg" style="text-align: center; height: 30px;" />
where:
<ol style="list-style-type: circle;">
	<li>hLminor is the head loss due to the component,</li>
	<li>KL is the loss coefficient specific to the component geometry,</li>
	<li>V is the fluid velocity</li>
	<li>g is the acceleration due to gravity.</li>
</ol>
For ease of calculation, engineers often represent minor losses from fittings and valves as an equivalent length of straight pipe that would produce the same head loss.
At the entry point of a pipe, sharp edges or abrupt changes in flow area can cause the fluid to separate from the wall, creating turbulence and energy dissipation through viscous effects. This entrance loss contributes to the overall energy loss, depending on the pipe geometry and the flow velocity. When fluid exits a pipe, the kinetic energy disperses into the surrounding environment, creating an exit loss characterized by a unity loss coefficient, as all the kinetic energy is lost from the system.
Sudden expansions in the pipe create high-velocity jets that decelerate as they expand, dissipating energy through viscous effects. These expansions lead to additional energy losses as the fluid experiences abrupt pressure and velocity changes. Bends in the pipe also contribute to head loss, primarily due to flow separation and swirling motions induced by centripetal forces as the fluid changes direction. The frictional resistance over the length of the bend adds further to the overall head loss, with the degree of loss depending on the bend angle and radius.
Understanding these minor losses allows engineers to design pipe systems that manage energy dissipation effectively, optimizing flow efficiency and minimizing pressure drops across the system.

Minor losses in pipe systems arise from components like valves, bends, and fittings that disrupt fluid flow, causing energy dissipation through turbulence ...

Major Losses in Pipes

When a fluid flows through a pipe, it experiences energy losses due to frictional resistance along the pipe walls, known as major losses. These energy losses result in a pressure drop, which varies based on the flow conditions &#8212; whether laminar or turbulent &#8212; and the specific physical properties of the fluid and pipe.
Fluid flow can be classified as laminar or turbulent, primarily based on the Reynolds number. This dimensionless number reflects the relative influence of inertial to viscous forces in the fluid. In laminar flow (Re &#60; 2000), the fluid flows in parallel layers, or streamlines, with minimal cross-stream mixing. Here, the pressure drop mostly depends on the fluid&#39;s viscosity and is generally lower. In contrast, turbulent flow (Re &#62; 4000) is characterized by chaotic eddies and swirling motions. In this regime, the pressure drop is influenced not only by viscosity but also by the roughness of the pipe wall, as these irregularities disrupt the flow further, increasing energy losses.
The pressure drop in a pipe depends on several factors: fluid properties, flow velocity, pipe characteristics, and dimensionless numbers.
The Darcy-Weisbach equation is the standard approach for quantifying the pressure drop due to frictional losses in pipe flow:
<img alt="Equation 1" src="/files/ftp_upload/18080/18080_Equation_1.svg" style="text-align: center; height: 30px;" />
Where:
<ol style="list-style-type: circle;">
	<li>&#916;P is the pressure drop,</li>
	<li>f is the friction factor,</li>
	<li>L is the length of the pipe,</li>
	<li>D is the pipe diameter,</li>
	<li>&#961; is the fluid density,</li>
	<li>v is the flow velocity.</li>
</ol>
The friction factor f is crucial for calculating pressure drops, especially in turbulent flow. It depends on the Reynolds number and the relative roughness of the pipe. Engineers often refer to the Moody chart, which provides empirical values of friction factors across flow regimes and roughness levels.
The Colebrook equation
<img alt="Equation 2" src="/files/ftp_upload/18080/18080_Equation_2.svg" style="text-align: center; height: 30px;" />
Where:
<ol style="list-style-type: circle;">
	<li>f is the friction factor,</li>
	<li>&#1013;/D is the relative roughness</li>
	<li>Re is the Reynolds number.</li>
</ol>
offers a precise method to calculate f for smooth and moderately rough pipes, but it is implicit and requires iterative solutions. To avoid iteration, approximations like the Haaland equation:
<img alt="Equation 3" src="/files/ftp_upload/18080/18080_Equation_3.svg" style="text-align: center; height: 30px;" />
Where:
<ol style="list-style-type: circle;">
	<li>f is the friction factor,</li>
	<li>&#1013;/D is the relative roughness</li>
	<li>Re is the Reynolds number.</li>
</ol>
are commonly used in practice, providing reasonably accurate friction factor values without extensive calculation. Understanding these dynamics enables engineers to design pipe systems that manage flow efficiently, compensating for potential pressure losses over time as pipe roughness increases.

Consider &#160;a fluid flowing through a pipe; it encounters frictional resistance, causing energy losses known as major losses, resulting in a pressure ...

Multiple Pipe Systems

Multipipe systems consist of complex configurations of interconnected pipes designed to transport fluids efficiently across intricate networks. They are essential in engineering applications requiring precise control over flow distribution, pressure, and head loss. They are categorized into series, parallel, loop, and network configurations, each distinguished by unique flow characteristics and applications.
Series Configuration
In a series configuration, fluid flows sequentially from one pipe segment to the next, maintaining a constant flow rate throughout. This setup means each segment experiences the same flow rate, although head losses accumulate progressively across each section. The total head loss from an entry point A to an exit point B is the cumulative sum of head losses in each pipe, governed by the Darcy-Weisbach equation for head loss or similar empirical relationships. This configuration suits applications requiring a consistent flow rate along a fixed path, such as in specific water treatment processes.
Parallel Configuration
Parallel multipipe systems divide fluid flow across multiple paths, enabling a higher overall flow rate and reducing resistance across the network. Here, the total flow rate is the sum of the individual flow rates in each branch. However, a critical feature of parallel systems is that all paths experience the same pressure drop. Consequently, fluid particles in each path encounter identical head losses, regardless of the path taken. This arrangement is effective in systems needing efficient fluid distribution, such as in heating and cooling systems, where multiple paths ensure temperature control across large areas.
Loop and Network Configurations
In loop systems, the flow rate within one pipe is balanced by the combined flow rates of the other connected pipes, enabling fluid redistribution based on demand changes or localized resistance variations, such as in urban water distribution systems. Network configurations represent the most complex setup, involving numerous interconnected pipes with multiple inlets and outlets,such as in oil and gas pipeline networks. This flexibility allows for multiple flow directions, enhancing transport efficiency and adaptability to dynamic system demands.
Applications of Multipipe Systems
Multipipe systems are indispensable in several engineering sectors. Municipal water supply networks often use network configurations to ensure consistent pressure and reliable delivery across urban areas. In deepwater oil and gas pipelines, multipipe systems help manage the challenging pressures and flow conditions of underwater environments. Additionally, industrial cooling systems benefit from the controlled fluid distribution provided by parallel configurations, which improve heat dissipation and maintain optimal temperatures in manufacturing processes.

Multiple pipe systems consist of interconnected pipes that regulate fluid flow across complex networks.
They can be arranged in series, parallel, loop, or ...

Turbulent Flow

Turbulent flow is characterized by unpredictable fluctuations in velocity and pressure, which result in a chaotic fluid movement distinct from the orderly patterns of laminar flow. While laminar flow is governed by smooth, parallel layers with minimal mixing, turbulent flow exhibits highly irregular, three-dimensional patterns. This behavior arises due to instabilities in the fluid&#39;s velocity profile, and amplifies as the flow velocity increases. Minor disturbances, known as turbulent spots, appear once the flow reaches a critical speed. When the Reynolds number &#8212; a dimensionless quantity expressing the ratio of inertial to viscous forces in a fluid &#8212; exceeds a threshold of around 4,000, these spots coalesce, generating fully developed turbulence.
Structure of Turbulent Flow in Pipes
In pipe systems, turbulent flow comprises three distinct regions: the viscous sublayer, the buffer layer, and the outer layer. The viscous sublayer forms close to the pipe wall, a thin boundary where viscous forces dominate, and the flow behaves similarly to laminar flow. Here, velocity follows a near-linear profile influenced by fluid viscosity, providing a stable region within the otherwise chaotic turbulent flow.
Above the viscous sublayer lies the buffer layer, a transitional zone where viscous and turbulent forces contribute to fluid motion. This region is marked by a logarithmic velocity profile that conforms to the logarithmic law of the wall, representing a balance between viscous and turbulent interactions. Further from the wall, turbulent eddies drive fluid movement in the outer layer, promoting rapid mixing and random motion across the flow.
Engineering Applications of Turbulent Flow
The chaotic nature of turbulent flow makes it advantageous in numerous engineering applications where mixing and energy dissipation are crucial. For instance, wastewater treatment systems rely on turbulent flow to enhance the mixing of chemicals, promoting efficient contaminant removal. In hydraulic engineering, turbulent flow dissipates energy in spillways, preventing structural damage by controlling water velocity. The turbulent mixing process also reduces river sediment buildup, maintaining navigable waterways and protecting aquatic ecosystems.

Turbulent flow is a fluid movement marked by chaotic shifts in pressure and velocity.
Unlike laminar flow, turbulent flow displays complex, irregular ...

Turbulent Flow: Problem Solving

Carbonation is a process used to dissolve carbon dioxide gas in a liquid, commonly used in the production of carbonated beverages. Achieving efficient carbonation requires careful control of temperature, pressure, and flow conditions. By adjusting these parameters, carbonation efficiency can be maximized, producing a higher concentration of CO2&#160;in the liquid.
Temperature is a key factor in CO2 solubility. In this case, the CO2 gas and the liquid are cooled to 20&#176;C. Lower temperatures enhance the solubility of CO2, enabling a greater volume of gas to dissolve within the liquid. At this temperature, the CO2&#160;gas is pressurized to approximately 550 kPa. Elevated pressure increases the partial pressure of CO2&#160;over the liquid, further driving dissolution.
CO2 is pumped through a connecting pipe between two tanks, where the flow characteristics are crucial for maintaining steady carbonation. The high Reynolds number, defined as greater than 4000, indicates that the flow is turbulent rather than laminar. Turbulent flow improves gas-liquid contact by promoting mixing, which enhances CO2&#160;dissolution in the liquid. The Reynolds number (Re) is calculated using the equation:
<img alt="Equation 1" src="/files/ftp_upload/18079/18079_Equation_1.svg" style="text-align: center; height: 30px;" />
where:
<ol style="list-style-type: circle;">
	<li>&#961; is the density of CO2,</li>
	<li>v is the velocity of flow,</li>
	<li>D is the pipe diameter,</li>
	<li>&#956; is the dynamic viscosity of CO2.</li>
</ol>
The flow rate of CO2&#160;in this system is controlled by the pipe&#39;s cross-sectional area and the velocity of the gas. To maintain turbulent conditions, the flow velocity v is adjusted based on the desired Reynolds number, which allows the required pipe diameter to be calculated.
The Reynolds number equation can be rearranged to determine the pipe diameter for the required turbulent flow. The&#160;dynamic viscosity &#956; of CO2&#160;at 20&#176;C are obtained using physical property tables. The optimal pipe diameter can be calculated by substituting these values along with the target Reynolds number and flow velocity into the equation. This configuration ensures efficient CO2&#160;transport through the system, optimizing the liquid&#39;s carbonation conditions.

Consider a liquid that needs to be carbonated. Both the liquid and the carbon dioxide gas are cooled. The temperature is selected as 20 degrees Celsius to ...

General Characteristics of Pipe Flow II

When fluid enters a pipe, it first passes through the entrance region, where the velocity profile adjusts due to viscous effects. In this region, a boundary layer forms along the pipe walls and grows until it fully occupies the pipe&#39;s cross-section. Once the boundary layer merges, the flow becomes fully developed, with a steady velocity profile that remains consistent along the pipe&#39;s length.
The distance to reach a fully developed flow is called the entrance length and depends on the flow regime (laminar or turbulent) and the Reynolds number. Laminar flow typically has a shorter entrance length due to its smooth, gradual profile development, whereas turbulent flow requires a longer entrance length due to enhanced mixing and chaotic motion.
A pressure difference along the pipe&#39;s length drives fully developed flow in a constant-diameter pipe. In horizontal pipes, this pressure difference overcomes viscous forces, ensuring steady motion. The entrance region has a higher pressure gradient to account for acceleration as the velocity profile evolves. Once fully developed, the pressure gradient stabilizes, reflecting the energy needed to overcome viscosity.
Shear stress behaves differently in laminar and turbulent flows. In laminar flow, shear stress arises from molecular momentum transfer, resulting in smooth, orderly layers. Turbulent flow stems from momentum exchange between more significant, irregularly moving fluid particles, making it a macroscopic effect. These differences influence pressure distribution and flow characteristics.
For inclined pipes, gravity modifies the pressure gradient. Depending on the pipe&#39;s orientation, gravity can assist or resist flow, altering the force balance required for steady motion. The viscosity, pressure, and gravity interplay determine fluid behavior within the pipe.

As fluid enters a pipe, it first flows through an entrance region where the velocity profile adjusts, forming a boundary layer along the walls. This ...

Laminar Flow

Laminar flow represents a smooth, orderly fluid motion where particles move along parallel paths, resulting in minimal mixing between layers. Streamlined particle paths characterize this flow regime and occur under conditions where viscous forces dominate over inertial forces. The distinction between laminar, transitional, and turbulent flow is primarily determined by the Reynolds number, a dimensionless quantity calculated as:
<img alt="Equation 1" src="/files/ftp_upload/18076/18076_Equation 1.svg" style="text-align: center; height: 30px;" />
where &#961; is the fluid density, v is the flow velocity, D is the pipe diameter, and &#956; is the dynamic viscosity. A Reynolds number below approximately 2100 indicates laminar flow, while values above 4000 denote turbulent flow. Transitional flow occurs between these ranges as the fluid shifts from laminar to turbulent.
Velocity Profile and Entrance Length
In a pipe, fluid initially entering develops a velocity profile over a length known as the entrance length. During this phase, the flow transitions from a uniform profile at the inlet to a fully developed parabolic profile, where the velocity is highest at the center and gradually decreases toward the pipe walls due to viscous forces. The entrance length is a function of the Reynolds number and pipe characteristics, typically shorter in turbulent flow and longer in laminar flow. Once fully developed, the parabolic velocity profile remains stable unless disrupted by changes in pipe geometry, allowing consistent analysis of flow behavior along the pipe&#39;s length.
Energy Loss and Hagen-Poiseuille Law
Laminar flow is advantageous in minimizing energy loss, as the pressure drop along the pipe is dominated by viscous forces rather than turbulence. The relationship between pressure drop and flow rate in a fully developed laminar flow is defined by the Hagen-Poiseuille equation:
<img alt="Equation 2" src="/files/ftp_upload/18076/18076_Equation 2.svg" style="text-align: center; height: 30px;" />
where &#916;P is the pressure drop, &#956; is the dynamic viscosity, L is the pipe length, Q is the volumetric flow rate, and R is the pipe radius. This equation shows that the pressure drop in laminar flow is directly proportional to flow rate and fluid viscosity and inversely proportional to the fourth power of the pipe radius, highlighting the significant impact of pipe diameter on energy efficiency.
Applications of Laminar Flow
Laminar flow conditions are essential in applications that require smooth, predictable fluid motion with minimal mixing. Wastewater treatment systems, for example, benefit from laminar flow to avoid excessive energy loss and facilitate controlled fluid handling. In canal designs for irrigation, laminar flow promotes even distribution without turbulent disturbances, enhancing water delivery efficiency. Hydropower systems can also leverage laminar flow for more efficient energy conversion, particularly in sections where consistent, low-energy flow is needed.

Laminar flow refers to the smooth, orderly motion of fluid particles in which each particle follows parallel paths with minimal mixing between layers.
The ...

Laminar Flow: Problem Solving

Laminar flow occurs when a fluid moves smoothly in parallel layers with minimal mixing and turbulence. In fluid mechanics, ensuring laminar flow within a pipe is essential for precise control of flow characteristics, especially in engineering applications. The key factor in determining whether flow remains laminar is the Reynolds number, a dimensionless quantity that depends on the fluid&#39;s velocity, density, viscosity, and the pipe&#39;s diameter. A Reynolds number of 2100 or lower indicates laminar flow, while higher values lead to turbulence.
For air flowing through a pipe at standard atmospheric conditions and a given mass flow rate, the velocity of the fluid must be controlled to maintain laminar conditions. Since velocity depends on the flow rate and the cross-sectional area of the pipe, the pipe diameter plays a crucial role. By substituting the velocity expression into the Reynolds number formula and solving for the diameter, it is possible to determine the minimum required pipe size.
Using standard values for air density and viscosity at 300 Kelvin, the volumetric flow rate can be computed. This, in turn, allows for the calculation of the necessary pipe diameter. To ensure a Reynolds number of 2100 or lower, the minimum allowable diameter for the pipe is found to be approximately 0.46 meters. This calculation ensures that the flow remains smooth and predictable, avoiding turbulence that could disrupt performance.

Consider air flowing through the pipe at 300 Kelvin at standard atmospheric pressure, with a mass flowrate of 0.0163 kilograms per second. If the flow is ...

General Characteristics of Pipe Flow I

Pipe flow refers to the movement of fluids within fully enclosed conduits, typically cylindrical in shape, such as water pipes or hydraulic hoses. These conduits are designed to withstand high-pressure gradients that drive fluid movement, contrasting with open-channel flows, where gravity is the primary driving force. Rectangular conduits, like air conditioning and heating ducts, generally operate at lower pressures and are less suited for high-pressure applications.
The classification of fluid flow in pipes was pioneered by Osborne Reynolds through his foundational dye injection experiments. Reynolds identified three flow regimes &#8212; laminar, transitional, and turbulent &#8212; based on fluid velocity. At low velocities, the dye maintained a smooth streakline, indicating laminar flow, where fluid particles follow orderly, parallel paths. As velocity increased, the dye began to oscillate, marking the transitional regime, and at higher velocities, it dispersed unpredictably in the turbulent regime, where random, chaotic motion dominates.
These observations led to the development of the Reynolds number, a dimensionless quantity that indicates the balance of inertial and viscous forces in the flow. The Reynolds number is calculated based on fluid velocity, density, viscosity, and pipe diameter and serves as an indicator of flow type: laminar for values below 2100, turbulent for values above 4000, and transitional in the range between. In laminar flow, velocity remains steady over time, while turbulent flow exhibits unsteady, fluctuating velocities with random components perpendicular to the pipe axis. By calculating the Reynolds number, engineers can predict flow behavior in pipe systems, aiding in the design and optimization of pipelines, hydraulic systems, and fluid transport networks across various industries.

Pipe flow describes fluid movement through enclosed, fully-filled conduits, typically cylindrical pipes like water pipes or hydraulic hoses, engineered to ...

Design Example: Creating a Hydraulic Model of a Dam Spillway

Scaled hydraulic models of dam spillways provide a practical way to replicate and study the intricate flow dynamics of these structures. Often built to a 1:15 ratio, these models allow for observing critical water behavior, such as velocity distribution, flow patterns, and energy dissipation.
<img alt="Equation 1" src="/files/ftp_upload/18074/18074_Equation_1.svg" style="text-align: center; height: 38px;" />
Through this controlled setup, researchers can closely examine how water flows over the spillway, supporting a detailed analysis of its performance. A fundamental principle behind these models is using Froude number similarity, which preserves the proper balance between gravitational and inertial forces.
<img alt="Equation 2" src="/files/ftp_upload/18074/18074_Equation_2.svg" style="text-align: center; height: 38px;" />
This similarity is essential, as it ensures that free-surface flow characteristics, including wave formation, turbulence, and flow separation, behave as they would in the full-sized structure. By achieving this accurate representation, scaled models provide valuable insights into how water interacts with spillway features, helping to pinpoint areas where the design might benefit from adjustments to enhance safety or flow efficiency.
In the scaled model, adjustments are also applied to discharge rates, flow velocities, and timing to reflect real-world conditions without requiring extensive water volumes or prolonged test durations. The discharge rate, for example, is reduced by the scale factor to maintain manageable flow rates, enabling safe experimentation.
<img alt="Equation 3" src="/files/ftp_upload/18074/18074_Equation_3.svg" style="text-align: center; height: 36px;" />
Additionally, time is compressed so that events unfolding over an entire day in the full-scale structure can be examined in just a few hours within the model.
<img alt="Equation 4" src="/files/ftp_upload/18074/18074_Equation_4.svg" style="text-align: center; height: 48px;" />
This efficient scaling supports rapid testing and analysis of different flow conditions and potential structural responses. Overall, scaled modeling of spillways is crucial for assessing designs, identifying potential vulnerabilities, and confirming that the full-scale structure will perform reliably under various water flow conditions.

A hydraulic model of a dam spillway is often created by scaling down the design, typically to a 1:15 ratio, to replicate key flow characteristics such as ...

Dimensionless Groups in Fluid Mechanics

Dimensionless groups in fluid mechanics provide simplified ratios that help analyze fluid behavior without relying on specific units. The Reynolds number (Re), which represents the ratio of inertial to viscous forces, distinguishes between laminar and turbulent flows, making it essential in the design of pipelines and aerodynamic surfaces. The Froude number (Fr), the ratio of inertial to gravitational forces, is particularly useful in predicting wave formation and hydraulic jumps in open-channel flows and coastal engineering.
The Euler number (Eu) indicates the ratio of pressure forces to inertial forces and is used to calculate pressure drops in systems such as pipelines and across valves. The Cauchy number (Ca), representing the ratio of inertial to elastic forces, is critical in compressible fluid flow analysis and the study of shockwaves. The Mach number (Ma) compares the flow speed to the speed of sound, making it crucial for studies in supersonic flight and aeroelasticity.
The Strouhal number (St), which compares oscillatory frequency to inertial forces, finds applications in resonance studies, particularly vortex shedding around structures like bridge piers. Lastly, the Weber number (We), showing the ratio of inertial to surface tension forces, is vital in analyzing droplet formation, spray dynamics, inkjet printing, and fuel atomization processes. These groups enable the comparison of fluid behaviors across different scales and systems, supporting a broad range of fluid dynamics applications.

The dimensionless groups in fluid mechanics are ratios of physical quantities used to describe and analyze fluid behavior independently of units of ...

Correlation of Experimental Data

Dimensional analysis simplifies complex physical problems and guides experimental investigations, but it does not provide complete solutions. It identifies the dimensionless groups that influence a phenomenon, but experimental data is needed to establish the specific relationships and validate theoretical predictions.
For example, a spherical particle moving through a viscous fluid experiences drag. Dimensional analysis shows that the drag force depends on the particle&#39;s diameter, velocity, and the fluid&#39;s viscosity. These variables are grouped into dimensionless terms to form a relationship, but experimental data is needed to determine the exact nature of this relationship.
Researchers measure drag force while varying velocity to correlate the dimensionless groups, such as the Reynolds number. This correlation allows the relationship between drag and velocity to be expressed as a constant. Multiple tests verify this constant, confirming the dependency of drag on velocity.
If drag depends only on diameter, velocity, and viscosity, the relationship should be universal for any spherical particle in any fluid under consistent conditions. Multiple tests with different particles and fluids verify this universality, reducing the need for further experiments, as the derived constant remains valid under the specified conditions.

A key use of dimensional analysis is to efficiently manage, interpret, and correlate experimental data.
Dimensional analysis alone cannot fully solve a ...

Typical Model Studies

Fluid mechanics model studies often utilize scaled-down systems to predict fluid behavior in full-scale environments, such as river flows, dam spillways, and structures interacting with open surfaces. Maintaining Froude number similarity in river models is crucial, as it replicates surface flow features like wave patterns and velocities.
<img alt="Equation 1" src="/files/ftp_upload/18073/18073_Equation_1.svg" style="text-align: center; height: 48px;" />
Practical constraints, however, can introduce geometric distortions, impacting how accurately these models represent the real system. Adjustments, such as increased model roughness, help bridge this gap, aligning flow behaviors between the model and the actual river environment.
For hydraulic structures like spillways, the focus shifts to achieving high Reynolds numbers to maintain dynamic similarity. This approach emphasizes balancing inertial and gravitational forces to replicate the prototype&#39;s behavior closely.
<img alt="Equation 2" src="/files/ftp_upload/18073/18073_Equation_2.svg" style="text-align: center; height: 45px;" />
Larger models are generally preferred, as they capture critical force interactions more effectively in full-scale designs. Although exact similarity across all scales may not be feasible due to physical and financial limitations, these larger models still allow close approximations of essential conditions.
Verification tests and prototype comparisons are key for refining model accuracy. Adjusting variables and calibrating outcomes ensure that the model reliably mirrors actual fluid flow behaviors. This predictive capability supports planning and design adaptations, making these models indispensable tools for assessing performance before moving to full-scale implementation.

Typical model studies analyze scaled-down systems to predict fluid behavior, focusing on interactions with free surfaces and flow around immersed bodies.
...

Modeling and Similitude

Scaled modeling is a fundamental technique in engineering, enabling the study of large and complex systems by creating smaller, manageable replicas that recreate critical characteristics of the original. In hydrology and civil infrastructure, for example, scaled models of dams help analyze water flow, turbulence, and pressure. This method allows for accurate predictions of real-world behavior within a controlled environment, significantly reducing the cost and time involved in full-scale testing, which is often impractical. These models provide valuable insights into system performance under various conditions by ensuring that essential design elements match.
For the model to yield valid insights, it must maintain similitude, meaning the replica accurately reflects the prototype&#39;s behavior through geometric, kinematic, and dynamic similarity. Geometric similarity ensures the scaled model retains the same proportional dimensions as the actual structure, while kinematic similarity requires that motion characteristics match between model and prototype. Dynamic similarity further demands that forces acting within the model accurately replicate those in the real system, accounting for any scaling effects.
In fluid dynamics, the Froude number is a key dimensionless parameter that matches scaled models to their full-sized counterparts, especially for applications like ship design. The Froude number is defined as:
<img alt="Equation 1" src="/files/ftp_upload/18072/18072_Equation_1.svg" style="text-align: center; height: 60px;" />
where V represents velocity, g is the gravitational acceleration, and L is a characteristic length, often the ship&#39;s length. By equating the Froude numbers for both the model and the full-scale ships, the gravitational and inertial forces align, allowing the scaled model to accurately predict behaviors such as maneuverability, stability, and wave formation.

A scaled model of a dam is tested in a controlled environment to observe how it will manage water flow, turbulence, and pressure.
This setup predicts the ...

Determination of Pi Terms

The Buckingham Pi theorem is a valuable method in dimensional analysis, reducing complex relationships between variables into dimensionless terms. Relevant variables in analyzing the lift force on an airplane wing include lift force, air density, wing area, aircraft velocity, and air viscosity. Expressing each variable in terms of fundamental dimensions &#8212; mass, length, and time &#8212; provides a consistent foundation for constructing these dimensionless terms.
The theorem indicates that the number of Pi terms equals the total variables minus the number of fundamental dimensions. In this example, five variables and three dimensions result in two Pi terms. Air density, velocity, and wing area are selected as repeating variables, as they independently cover all dimensions. These repeating variables combine with lift force and viscosity to form dimensionless Pi terms.
<img alt="Equation 1" src="/files/ftp_upload/18069/18069_Equation_1.svg" style="text-align: center; height: 100px;" />
The first Pi term expresses the efficiency of lift generation, representing how lift depends on density, velocity, and area. The second Pi term accounts for the relative impact of viscosity, revealing how viscous forces interact with the wing in relation to inertia.
Each Pi term is verified as dimensionless, allowing complex aerodynamic behavior to be represented in simplified expressions. This approach enables the prediction and optimization of lift under various conditions, capturing the essential dynamics without needing to examine each factor individually.

To determine Pi terms using the Buckingham Pi theorem, consider the example of analyzing lift force on an airplane wing.
Relevant variables here include ...

Dimensional Analysis

Dimensional analysis is a valuable technique in fluid mechanics for simplifying complex problems by reducing them into dimensionless groups. These groups capture the essential relationships between the variables involved, allowing researchers and engineers to analyze fluid flow without dealing with each variable individually. This approach reduces the number of independent variables, allowing for easier analysis and better understanding of physical phenomena.
In fluid mechanics, dimensional analysis is used to understand the behavior of fluids in systems such as pipelines, channels, and other structures common in civil engineering. Consider the example of fluid flow through a long, smooth-walled, horizontal circular pipe, which is typical in the design of water distribution systems or sewage networks. The pressure drop per unit length along the pipe depends on variables such as pipe diameter, fluid density, fluid velocity, and viscosity. According to dimensional analysis, the pressure drop can be expressed as a function of these variables:
<img alt="Equation 1" src="/files/ftp_upload/18067/18067_Equation_1.svg" style="text-align: center; height: 12px;" />
This formulation involves five independent variables, which can complicate the analysis. To simplify this, we can use dimensional analysis to reduce the number of variables into two dimensionless groups, thus streamlining the problem. Two important dimensionless groups that emerge from this analysis are the Reynolds number, which characterizes the flow regime, and the friction factor, which correlates with the pressure drop.
<img alt="Equation 2" src="/files/ftp_upload/18067/18067_Equation_2.svg" style="text-align: center; height: 30px;" />
Engineers use the dimensionless groups to generate a universal curve that applies to any smooth-walled pipe and incompressible Newtonian fluid. This curve would allow them to predict the pressure drop for different pipe sizes and fluids without performing numerous experiments for each possible combination.
<img alt="Figure 1" src="/files/ftp_upload/18067/18067_PT_Figure.svg" style="text-align: center; height: 250px;" />
This approach minimizes the time and cost required for experimentation and allows for more straightforward design decisions. Whether designing water distribution networks or analyzing pressure losses in irrigation systems, dimensional analysis is critical for any civil engineer.

Dimensional analysis helps simplify fluid flow problems by using dimensionless groups, reducing complex variables into simpler terms that are easier to ...

The Buckingham Pi Theorem

The Buckingham Pi theorem provides a structured method to simplify fluid dynamics problems by reducing complex systems of variables to dimensionless terms.
<img alt="Equation 1" src="/files/ftp_upload/18068/18068_Equation_1.svg" style="text-align: center; height: 34px;" />
In analyzing flow through a pipe, variables that define the system include the pipe&#39;s diameter, the fluid&#39;s velocity, its density, and viscosity. These can be combined into a single dimensionless term, the Reynolds number (Re), which captures their essential relationship.
<img alt="Equation 2" src="/files/ftp_upload/18068/18068_Equation_2.svg" style="text-align: center; height: 38px;" />
The first step in applying the Buckingham Pi theorem is identifying the fundamental dimensions of each variable. In fluid mechanics, these are typically mass, length, and time. Here, the diameter represents length, velocity combines length and time, density encompasses mass and volume, and viscosity incorporates mass, length, and time.
<img alt="Equation 3" src="/files/ftp_upload/18068/18068_Equation_3.svg" style="text-align: center; height: 140px;" />
The theorem suggests that the number of independent dimensionless groups, known as Pi terms, is the difference in the number of variables k and the number of fundamental dimensions r. Only one dimensionless term is needed with four variables and three dimensions, simplifying analysis.
<img alt="Equation 4" src="/files/ftp_upload/18068/18068_Equation_4.svg" style="text-align: center; height: 32px;" />
This single term, the Reynolds number, compares inertial forces to viscous forces in the fluid. Establishing this ratio reveals whether the flow is likely to be smooth and orderly, known as laminar flow, or chaotic and irregular, known as turbulent flow. Lower values indicate laminar flow, where viscous forces dominate, and the fluid moves in parallel layers. Higher values reflect turbulent flow, where inertial forces lead to chaotic motion. The Buckingham Pi theorem&#39;s application in this context reduces complex interactions to a single term, making it easier to generalize flow behavior under varying conditions.

Consider the flow through a pipe, where key variables include the pipe&#39;s diameter D, fluid velocity V, density &#961;, and viscosity &#956;.
To ...

Navier&#8211;Stokes Equations

For incompressible Newtonian fluids, where density remains constant, stresses show a linear relationship with the deformation rate, defined by normal and shear stresses. Normal stresses depend on the pressure exerted on the fluid and the rate of deformation in specific directions, which determines how fluid flows under varying pressures. Shear stresses, on the other hand, act tangentially across fluid layers. They explain how adjacent fluid layers slide relative to one another, connecting tangential stress to velocity differences between layers. Normal and shear stresses define the internal forces within a fluid that influence its movement.
The Navier-Stokes equations are derived by incorporating these stress relationships into differential equations of motion.
<img alt="Equation 1" src="/files/ftp_upload/18062/18062_Equation_1.svg" style="text-align: center; height: 106px;" />
These equations balance inertial, pressure, and gravitational forces in a viscous fluid and are fundamental to predicting fluid behavior under various conditions. The inertia term in the Navier-Stokes equations accounts for fluid acceleration, representing the fluid&#39;s resistance to changes in velocity. This term is critical in capturing the momentum conservation of a moving fluid, as it demonstrates how fluids resist sudden shifts in speed or direction.
The pressure gradient term within the Navier-Stokes equations drives fluid movement by creating a net force from areas of high pressure to low pressure. Meanwhile, the viscous terms represent internal friction arising from molecular interactions within the fluid. Depending on the velocity gradients present, these viscous forces act to slow down the fluid movement.
Each directional Navier-Stokes equation captures internal forces like viscous stress and external forces like gravity. These equations allow for predicting fluid motion in various conditions, from simple to complex flows. For steady or laminar flow scenarios, where flow is smooth and orderly, the equations become more manageable, enabling more straightforward analysis. This simplification is useful in controlled scenarios like boundary layer flow, where flow near a surface has a structured layer, and in Couette flow, describing fluid between two parallel surfaces moving at different velocities.

In fluid motion under viscous conditions, shear stress is directly proportional to fluid deformation. In incompressible Newtonian fluids, this stress ...

Plane Potential Flows

Plane potential flows simplify fluid motion by assuming the fluid to be irrotational and incompressible. These characteristics allow these flows to be described by a velocity potential function, &#981;, representing the flow speed in a given direction, and a stream function, &#968;, that visualizes the flow path, both governed by Laplace&#39;s equation. These parameters help in estimating flow patterns, velocity distributions, and pressure fields around various hydraulic structures.
Uniform Flow
Uniform flow represents the simplest form of potential flow, with fluid moving in a straight line at a constant velocity. Streamlines are straight, parallel, and evenly spaced, clearly visualizing flow across channels or pipes with constant velocity. This flow type is essential in designing canals, pipelines and drainage systems, where the assumption of constant velocity aids in flow rate estimation and hydraulic design.
Source and Sink Flows
Source and sink flows describe radially symmetric flows originating from or converging to a point. In a source flow, fluid moves radially outward from a central point, while in a sink flow, it converges inward. These flows are modeled using a flow rate parameter to represent the strength of the source or sink. This model is widely used in groundwater hydrology, where source flows represent groundwater recharge, such as that from an injection well, and sink flows represent groundwater extraction in a pumping well.&#160; represent groundwater extraction from a pumping well., and sink flows simulate groundwater recharge .
Vortex Flow
Vortex flow involves fluid circulating a central point and appears in two forms: free and forced. In a free vortex, particles move in circular paths around a center point without external force, creating a whirlpool effect often observed in natural bodies of water. This model is used to design spillways and drainage systems that manage spiraling water flow to prevent clogging or turbulence. In a forced vortex, an external force drives the fluid&#39;s rotation, creating a scenario where the fluid rotates as a rigid body. Forced vortices are used in sedimentation tanks and certain hydraulic devices where uniform rotation aids in sediment separation and fluid mixing.
Flow Nets
In flow nets, streamlines represent the fluid flow path, while equipotential lines mark points of equal hydraulic potential. These lines intersect perpendicularly, creating a grid pattern that aids in identifying pressure distributions and potential seepage paths around structures. Flow nets are constructed to assess seepage in soil or porous media. Seepage rates are estimated by carefully analyzing the spacing and orientation of these lines and identifying potential areas of erosion or structural weakness.

Plane potential flows simplify fluid motion by assuming irrotational and incompressible flow, governed by a velocity potential function and a stream ...

Steady, Laminar Flow Between Parallel Plates

Understanding steady, laminar flow between parallel plates is essential for analyzing and designing flow in narrow rectangular channels, commonly found in various water conveyance and drainage systems. The Navier-Stokes equations govern fluid motion and are generally challenging to solve due to their nonlinearity. However, simplifications are possible in certain cases, like the steady laminar flow between parallel plates. For this scenario, we assume steady, incompressible, laminar flow.
Considering two horizontal plates aligned along the x-axis and spaced a distance &#39;2h&#39; apart, with the y-axis perpendicular to the plates, fluid motion is assumed to be parallel. Here, the continuity equation indicates no change in velocity along the x-axis, and the Navier-Stokes equations reduce as follows:
<img alt="Equation 1" src="/files/ftp_upload/18063/18063_Equation_1.svg" style="text-align: center; height: 30px;" />
The equations in the y and z directions show no pressure gradient along these axes, indicating that pressure varies hydrostatically in the y-direction, allowing integration to yield:
<img alt="Equation 2" src="/files/ftp_upload/18063/18063_Equation_2.svg" style="text-align: center; height: 12px;" />
For parallel plates with a pressure gradient along the x-axis, the equation simplifies:
<img alt="Equation 3" src="/files/ftp_upload/18063/18063_Equation_3.svg" style="text-align: center; height: 30px;" />
By integrating twice and applying boundary conditions (u = 0 at y = +h and y = -h), we obtain:
<img alt="Equation 4" src="/files/ftp_upload/18063/18063_Equation_4.svg" style="text-align: center; height: 30px;" />
This expression reveals that the velocity profile across the gap between the plates is parabolic, with maximum velocity at y = 0, midway between the plates. The volumetric flow rate through a unit width of the plates is obtained by integrating the velocity profile across the gap.
Understanding laminar flow between parallel plates has practical applications in groundwater flow, wastewater treatment, and lubrication. For instance, this model can approximate flow in soil layers, where water moves between soil particles like flow between plates. Knowing the flow rate and velocity distribution helps engineers design efficient drainage systems and predict groundwater movement.

Steady, laminar flow between two parallel plates describes fluid movement in a narrow, confined channel, often used to model simple fluid systems.
For ...

Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The overall function of the immune system is to defend the body against infectious organisms and other invaders. White blood cells, or leukocytes, are the key players of the immune system. Upon infection, they are activated and initiate an immune response. Leukocytes can be divided into various sub-populations (e.g., myeloid cells, lymphocytes, dendritic cells) based on different parameters that can be biological, physical, and/or functional (e.g., size, granularity, and secretion). One way to characterize leukocytes is through their surface proteins, which are mainly receptors. Each leukocyte population expresses a specific combination of receptors (e.g., cytotoxic, activating, migration receptors) that can define subsets among populations. As the immune system encompasses a wide range of cell populations, it is essential to characterize them to decipher their participation in the immune response.
Flow cytometry (FC or FCM) is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogenous cell mixture. Flow cytometers are composed of three main sub-systems: fluidics, optics, and electronics. The fluidics system transports the cells in a stream such that they pass in front of a laser one by one. The optics system consists of light sources (lasers) to illuminate the particles, optical filters to direct the resulting light, and fluorescent signals to appropriate detectors. Finally, the electronics system converts the detected light signals into electronic signals that can be processed by the computer. As an individual cell passes in front of the laser beam, it scatters light. A detector in front of the beam measures forward scatter (FS) and several detectors to the side measure side scatter (SC). The FS correlates with cell size and SC is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
In addition to analyzing a cell&#39;s size, shape, and complexity, flow cytometry is widely used for detecting the expression of cell surface receptors (1). This is accomplished by using fluorochrome-labelled monoclonal antibodies which bind to known cell-specific receptors. Upon excitation, these bound fluorochromes emit a light of specific wavelength, called emission wavelength, which can be detected and scored. Fluorescence measurements provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors. Hematologists were first to use FC for the therapeutic follow up of immune cell populations (2). Now, it is used for a wide range of applications such as immunophenotyping, cell viability, gene expression, cell counting, and GFP analysis.
FACS (Fluorescent Activated Cell Sorter) is a specialized type of flow cytometry, which sorts a population of cells into subpopulation using fluorescent labelling. Just like conventional flow cytometry, first FS, SC, and fluorescent data are collected. Then, the machine applies a charge (negative or positive) and an electrostatic deflection system (electromagnets) facilitates the collection of charged droplets containing cells into appropriate tubes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10494/10494fig1.jpg" /> 
Figure 1: Schematic representation of FACS. Sample (1) is aspirated in the FACS (2) and passed in front of the laser (3). Cell fluorescence is sensed by fluorescence detectors (4). Finally, cells are incorporated in droplets and the cells of interest are deflected by deflection plates (5) and collected in a collection tube (6). The remaining cells go into the trash (7). <a href="https://www.jove.com/files/ftp_upload/10494/10494fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sorting aspect of the FACS presents many advantages. Many tests can help understand the role of specific cells in immune system, such as analyses of gene expression like RT-qPCR, cell cycle, or cytokine secretion. However, cells should be purified upstream to obtain clear and specific results. Here, FACS comes in useful and the desired cells can be sorted with great purity, yielding highly reliable and reproducible results. FACS can also be used to sort cells based on nuclear or other intracellular staining and according to the presence, absence, and density of surface receptors. FACS is now a standard technique for the purification of subpopulations of cells and has the ability sort up to four populations simultaneously.
This lab exercise demonstrates how to isolate splenic leukocytes and then how to specifically sort B lymphoid cells from the splenic leukocyte cell mixture using FACS.

Flow Cytometry and FACS: Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Defence against pathogens depends on surveillance by the immune system. This system is complex and comprises many cell types, each one with specific functions. This complex composition enables immune responses to a large diversity of pathogens and injuries. Adaptive immunity allows specific responses against specific pathogens. The majority of cells responsible for this type of immunity are the lymphocytes (B cells and T cells). Usually, B cells respond to extracellular infections (such as bacterial infections), and T cells respond to intracellular infections (such as viral infections). The different types of cells in lymphocyte populations can be characterized by the combination of cell surface proteins they express and/or by a panel of secreted cytokines.
Magnetic sorting allows enrichment of targeted cell populations using magnetic properties and expression of one or several cell surface proteins (1, 2). This technique consists of three steps. First, the cells are incubated with magnetic beads that are coupled with one or several monoclonal-specific antibodies. Cells that express surface proteins that bind to these antibodies attach to the magnetic beads. Then, the targeted cell populations are captured with a magnet. To finish, the targeted cells are eluted from the magnet. At the end, two sorting products are obtained, one containing unlabeled cells and the second containing the target cells coupled with the magnetic beads. Columns can be used to improve the efficiency of magnetic sorting. In the column, a non-magnetic element lengthens the path of cell through the column. Hence, cell flow is slowed down, facilitating cell capture by the magnet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10495/10495fig1.jpg" /> 
Figure 1: Schematic representation of magnetic separation. Thymic leukocytes are stained with anti-CD3 biotinylated antibodies. After washing, streptavidin (SAV) coupled beads specifically fix the biotin on anti-CD3 antibodies. (1) Cells are transferred in a column. (2) The magnet does not retain unlabeled cells, while CD3-positive cells remain in the column. Finally, the column is separated from the magnet and (3) CD3-positive cells are eluted in medium. <a href="https://www.jove.com/files/ftp_upload/10495/10495fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are two types of magnetic sorting (3). In positive sorting, cells of interest are captured with the magnetic beads. In negative sorting, unwanted cells are removed by capturing with the magnetic beads carrying the appropriate antibodies. This MACS technique permits good enrichment of targeted cells and improves the percentage of recovered cells from 1-20% to 60-98% in an organ. After sorting, it is necessary to verify cell purity and sorting by different methods (e.g. flow cytometry). The MACS technique is ideal to enrich a target population for other experiments such as cell culture or cell cycle analysis.
In this lab exercise, we demonstrate how to isolate thymic leukocytes and thereafter enrich thymic CD3-positive cells from the mix using magnetic cell sorting technique.

MACS: Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

ELISA Assays: Indirect, Sandwich, and Competitive

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4 
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Enzyme-linked immunosorbent assay (ELISA) is frequently used to measure the presence and/or concentration of an antigen, antibody, peptide, protein, hormone, or other biomolecule in a biological sample. It is extremely sensitive, capable of detecting low antigen concentrations. The sensitivity of ELISA is attributed to its ability to detect the interactions between a single antigen-antibody complex (1). Moreover, the inclusion of an enzyme-conjugated antigen-specific antibody permits the conversion of a colorless substrate into a chromogenic or fluorescent product that can be detected and easily quantitated by a plate reader. When compared to the values generated by titrated amounts of a known antigen of interest, the concentration of the same antigen in the experimental samples can be determined. Different ELISA protocols have been adapted to measure antigen concentrations in a variety of experimental samples, but they all have the same basic concept (2). Choosing the type of ELISA to perform, indirect, sandwich, or competitive, depends on a number of factors, including the complexity of the samples to be tested and the antigen-specific antibodies available to use. The indirect ELISA is frequently used to determine the outcome of an immunological response, such as measuring the concentration of an antibody in a sample. The sandwich ELISA is best suited for analyzing complex samples, such as tissue culture supernatants or tissue lysates, where the analyte, or antigen of interest, is part of a mixed sample. Finally, the competitive ELISA is most often used when there is only one antibody available to detect the antigen of interest. Competitive ELISAs are also useful for detecting a small antigen with only a single antibody epitope that cannot accommodate two different antibodies due to steric hinderance. The protocol will describe the basic procedures for the indirect, sandwich, and competitive ELISA assays.
The indirect ELISA assay is commonly used to measure the amount of antibodies in serum or in the supernatant of a hybridoma culture. The general procedure for the indirect ELISA assay is:
<ol>
	<li>Coat wells with antigens</li>
	<li>Add serum or hybridoma culture supernatant containing antibody (primary or 1&#176; antibody)</li>
	<li>Incubate and wash</li>
	<li>Add secondary (or 2&#176;) enzyme-conjugated antibody</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
The sandwich ELISA assay differs from the indirect ELISA assay in that the method does not involve coating the plates with a purified antigen. Instead, a &#34;capture&#34; antibody is used to coat the wells of the plate. The antigen is &#34;sandwiched&#34; between the capture antibody and a second &#34;detection&#34; enzyme-conjugated antibody - where both antibodies are specific for the same antigen, but at different epitopes (3). By binding to the capture antibody/antigen complex, the detection antibody remains in the plate. Either monoclonal antibodies or polyclonal antisera can be used as the capture and detection antibodies. The main advantage of the sandwich ELISA is that the sample does not have to be purified before analysis. Moreover, the assay can be quite sensitive (4). Many commercially available ELISA kits are of the sandwich variety and use tested, matched pairs of antibodies. The general procedure for the sandwich ELISA assay is:
<ol>
	<li>Coat wells with capture antibody</li>
	<li>Add test samples containing antigen</li>
	<li>Incubate and wash</li>
	<li>Add enzyme-conjugated detection antibody.</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
Most commercially available sandwich ELISA kits come with enzyme-conjugated detection antibodies. In cases where an enzyme-conjugated detection antibody is not available, a secondary enzyme-conjugated antibody specific for the detection antibody can be used. The enzyme on the secondary antibody performs the same role, which is to convert the colorless substrate to a chromogenic or fluorescent product. The above-mentioned secondary enzyme-conjugated antibody would more like to be used in a &#34;homemade&#34; sandwich ELISA developed by an investigator who has generated their own monoclonal antibodies, for example. One drawback to using a secondary enzyme-conjugated antibody is to be sure it only binds to the detection antibody, and not the capture antibody bound to the plate. This would result in a measurable product in all wells, regardless of the presence or absence of antigen or detection antibody.
Finally, the competitive ELISA assay is used to detect soluble antigens. It is simple to perform, but it is only suitable when the purified antigen is available in a relatively large amount. The general procedure for the competitive ELISA assay is:
<ol>
	<li>Coat wells with antigen</li>
	<li>Incubate and wash</li>
	<li>Preincubate test sample with enzyme-conjugated primary antibodies</li>
	<li>Add mixture to well</li>
	<li>Incubate and wash away any unbound enzyme-conjugated primary antibody</li>
	<li>Add substrate</li>
</ol>
The &#34;competition&#34; in this assay comes from the fact that more antigen in the test sample used in step 3 will result in less antibody available to bind to the antigen coating the well. Thus, the intensity of the chromogenic/fluorogenic product in the well at the end of the assay is inversely related to the amount of antigen present in the test sample.
A key component in any type of ELISA is the titrated standards of known concentrations that will allow the user to determine the antigen concentration present in the test samples. Typically, a series of wells are designated for creating a standard curve, where known amounts of a purified recombinant protein are added to the wells in decreasing amounts. When these wells are processed at the same time as the test samples, the user can then have a reference set of absorbance values obtained from a microplate reader for known protein concentrations to go along with the absorbance values for the test samples. The user can then calculate a standard curve to which the test samples can be compared for determining the amount of protein of interest present. The standard curve can also determine the degree of precision of the user&#39;s dilution making.
Finally, the last step in each of the ELISA types listed above calls for the addition of a substrate. The degree of conversion of the substrate to product is directly related to the amount of enzyme present in the well. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes found conjugated to antibodies. As expected, there are a number of substrates available specific for either enzyme that produce a chromogenic or fluorescent product. Moreover, substrates are available in a range of sensitivities that can increase the overall sensitivity of the assay. The user must also take into consideration the type of instrumentation available for reading the plate at the end of the experiment when picking the type of substrate to use, along with its corresponding enzyme-conjugated antibody.
Commonly used chromogenic substrates for HRP include 2,2&#39;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) and 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB), whereas p-Nitrophenyl Phosphate (PNPP) is used for AP. ABTS and TMB produce water-soluble green and blue colored reaction products, respectively. The green ABTS product has two major absorbance peaks, 410 and 650 nm, while the blue TMB product is best detected at 370 and 652 nm. The colors of ABTS and TMB change to yellow upon the addition of an acidic stop solution, which is best read at 450 nm. Color development for ABTS is slow, while it is fast for TMB. TMB is more sensitive than ABTS, and may produce a higher background signal if the enzymatic reaction proceeds too long. PNPP produces a yellow water-soluble product after AP conversion that absorbs light at 405 nm.

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455
...

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

Source: Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed following pathogen re-exposure, thus it is critical to be able to detect these cells. Therefore, to clearly assess antigen-specific memory B-cell responses, both ELISA and ELISPOT should be used (2).
ELISPOT assay uses a plate containing membrane-lined wells that are coated with antibodies in order to capture secreted proteins of interest. Then, the plate is loaded with cells and stimuli to induce protein production. The secreted proteins are captured by the antibodies coated on the surface. After appropriate incubation time, cells are removed and the secreted molecule is detected by using a biotinylated antibody that is specific for a different epitope, as compared to the capture antibody. Next, streptavidin peroxidase is added, followed by the addition of a substrate that allows the detection of the spots (Figure 1). The strength of this assay is that it allows one to quantitate the number of cells producing the protein of interest. Importantly, one can assess if there are changes in the total number of cells producing a specific protein or if individual cells within a population are producing more protein. Moreover, it can provide information regarding kinetics and can be used to assess overall immune activation (mitogen stimulation) relative to antigen-specific responses (antigen simulation). The ELISPOT assay will permit the detection one activated cell amongst 300,000 cells following mitogenic or antigen-specific activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" /> 
Figure 1: ELISPOT protocol overview.
The major advantages of this assay are its- a. Simplicity- the protocol is relatively simple and straightforward. It does not require technical expertise, b. Sensitivity- it permits the detection of immune cells at the single cell level and requires very few cells compared to other methods such as flow cytometry, c. Functionality- it provides quantitative data regarding immune function.
This lab exercise demonstrates the ELISPOT protocol for detection of IFN-&#947; secreting-splenocytes, but as mentioned above this assay can also be used to assess antibody secretion by B cells (3).

ELISPOT Assay: Detection of IFN-&amp;#947; Secreting Splenocytes

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://www.jove.com/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 
1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 
2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 
4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455
Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue structure and contribute to our understanding of mechanisms of disease. Tissue biopsies, either from patients or from experimental in vivo models, are often preserved by fixing in formalin or paraformaldehyde and embedding in paraffin wax. This allows for long-term storage and for the tissues to be sectioned. Tissues are cut into thin (5 &#181;m) sections using a microtome and the sections are adhered to glass slides. The tissues sections can be stained with antibodies, which allow for the detection of specific proteins within the tissue sections. Staining with antibodies conjugated to fluorophores (also known as fluorochromes) - compounds that emit light at specific wavelengths when excited by a laser - is known as immunofluorescence. The ability to detect proteins within a section can provide information such as cell type heterogeneity within the tissue, activation of specific signaling pathways, and expression of biomarkers. Depending on the fluorophores used and type of microscope available for analysis, multiple colors can be used, which allows for multiplexed analysis of targets.
The following protocol outlines the basic steps involved in immunofluorescence staining of paraffin embedded tissue sections. It is important to note that this protocol will not include any details on the fixation of tissue, process of paraffin embedding, or sectioning of the tissues. Once tissues have been sectioned and placed on glass slides, they are rehydrated through a series of graded ethanol (EtOH) incubations. The sections are incubated with a blocking reagent to reduce non-specific binding of antibody to the tissue section. The sections are then incubated with a primary antibody that may or may not be directly labeled with a fluorophore. If the primary antibody is not directly labeled, the sections are then incubated with a secondary antibody labeled with a fluorophore. Different antibodies may require different staining conditions, thus suggestions for optimization of antibodies are included. Following washing to remove all unbound antibody, the slides are mounted with media containing DAPI to fluorescently label the nucleus. Once the mounting media has dried, the slides can be imaged using a microscope with lasers that can detect the different fluorophores.

Immunofluorescence Staining and Microscopy

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4
1 Department of Lab Medicine and Pathology, University of Minnesota, ...

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 
2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional &#34;wide-field&#34; epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through &#34;laser scanning&#34;- typically a set of voltage-controlled mirrors (galvanometer or &#34;galvo&#34; mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software.
Before discussing the mechanism of a confocal microscope, it is important to consider how a sample interacts with light. Light is composed of photons, packets of electromagnetic energy. A photon impinging on a biological sample can interact with the molecules comprising the sample in one of four ways: 1) the photon does not interact and passes through the sample; 2) the photon is reflected/scattered; 3) the photon is absorbed by a molecule and the absorbed energy is released as heat through processes collectively known as nonradiative decay; and 4) the photon is absorbed and the energy is then rapidly reemitted as a secondary photon through the process known as fluorescence. A molecule whose structure permits fluorescence emission is called a fluorophore. Most biological samples contain negligible endogenous fluorophores; therefore exogenous fluorophores must be used to highlight features of interest in the sample. During fluorescence microscopy, the sample is illuminated with light of the appropriate wavelength for absorption by the fluorophore. Upon absorbing a photon, a fluorophore is said to be &#34;excited&#34; and the process of absorption is referred to as &#34;excitation&#34;. When a fluorophore gives up energy in the form of a photon, the process is known as &#34;emission&#34;, and the emitted photon is called fluorescence.
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a &#34;focal point&#34; where it is maximally focused. Beyond the focal point the light again diverges. The entering and exiting beams may be visualized as a pair of cones touching at the focal point (see Figure 1, left panel). The phenomenon of diffraction imposes a limit on how tightly a beam of light can be focused - the beam actually focuses to a spot of finite size. Two factors determine the size of the focal spot: 1) the wavelength of the light, and 2) the light-gathering ability of the objective lens, which is characterized by its numerical aperture (NA). The focal &#34;spot&#34; extends not only in the x-y plane, but also in the z direction, and is in reality a focal volume. The dimensions of this focal volume define the maximum resolution achievable by optical imaging. Although the number of photons is greatest within the focal volume, the conical light paths above and below the focus also contain a lower density of photons. Any fluorophore in the light path can thus be excited. In conventional (wide-field) epifluorescence microscopy, emission from fluorophores above and below the focal plane contribute out-of-focus fluorescence (a &#34;hazy background&#34;), which reduces image resolution and contrast, as demonstrated in Figure 1, with the red cube representing fluorophore emission above the focal plane (red star) that results in out-of-focus fluorescence (top right). This problem is ameliorated in confocal microscopy, due to the utilization of a pinhole. (Figure 2, bottom right). As depicted in Figure 3, the pinhole allows emissions originating from the focal place to reach the detector (left), while blocking the out-of-focus fluorescence (right) from reaching the detector, thus improving both resolution and contrast.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig1.jpg" /> 
Figure 1. Optical resolution of epifluorescence versus confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a focal volume and then diverges (left). The red star represents the focal plane of a sample that is being imaged while the red square represents fluorophore emission above the focal plane. When capturing an image of this sample using an epifluorescent microscope, the emission from the out-of-focus red square will visible and contribute to a &#34;hazy background&#34; (top right). Confocal microscopes have a pinhole which prevents the detection of light emitted outside of the focal plane, eliminating the &#34;hazy background&#34; (bottom right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig2.jpg" /> 
Figure 2. Pinhole effect in confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the highest intensity of the excitation light is at the focal point of the lens (left, red oval), other parts of the sample not in the focal point (right, red star) will get light and fluoresce. To prevent light emitted from these out-of-focus regions to reach the detector, a screen with a pinhole is present in front of the detector. Only the in-focus light (left) emitting from the focal plane is able to travel through the pinhole and reach the detector. The out-of-focus light (right) is blocked with the pinhole and fails to reach the detector.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig3.jpg" /> 
Figure 3. Principal components of a confocal laser scanning microscope. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the sake of simplicity, the mechanistic description of a confocal microscope will be limited to that of the Nikon Eclipse Ti A1R. Although there may be minor technical differences between different confocal microscopes, the A1R serves well as a good model for describing confocal microscope function. The excitation light beam, produced by an array of diode lasers, is reflected by the primary dichroic mirror into the objective, which focuses the light onto the specimen being imaged. The primary dichroic mirror selectively reflects the excitation light while allowing light at other wavelengths to pass through. The light then encounters the scanning mirrors that sweep the light beam across the specimen in an x-y manner, illuminating a single (x,y) pixel at a time. Fluorescence emitted by fluorophores at the illuminated pixel is collected by the objective lens and passes through the primary dichroic mirror to reach an array of photomultiplier tubes (PMTs). Secondary dichroic mirrors direct the emission light to the appropriate PMT. Excitation light scattered by the sample back into the objective is reflected by the primary dichroic mirror back towards the specimen, and thus prevented from entering the detection light path and reaching the PMTs (see Figure 3). This allows the relatively feeble fluorescence to be quantified without contamination by light scattered from the excitation light beam, which is typically orders of magnitude more intense than the fluorescence. Because the pinhole blocks light from outside of the focal volume, the light arriving at the detector comes from a narrow, selected z-plane. Therefore, images can be collected from a series of adjacent z-planes; this series of images is often referred to as a &#39;z-stack&#39;. By using the appropriate software, a z-stack can be processed to generate a 3D image of the specimen. A particular advantage of confocal microscopy is the ability to distinguish the subcellular localization of staining. For example, the differentiation between membrane staining from intracellular staining, which is very challenging with conventional epifluorescence microscopy (1, 2, 3).
Sample preparation is an important facet of confocal imaging. A strength of optical microscopy techniques is the flexibility to image live or fixed cells. When attempting to produce 3D images, because of the number of images that must be acquired for a z-stack, the difficulty of maintaining cell health, and the movement of live cells and their organelles, the use of fixed cells is typical. The procedure for fixing and staining cells for confocal fluorescence is similar to that conventionally used in immunofluorescence. After culture in chamber slides or on coverslips, cells are fixed using paraformaldehyde to preserve cellular morphology. Non-specific antibody binding is blocked using bovine serum albumin, milk, or normal serum. In order to maintain the specificity of the secondary antibodies, the solution used should not originate from the same species in which the primary antibodies were generated. The cells are incubated with primary antibodies that bind the antigen of interest. When labeling several cellular targets, the primary antibodies must each be derived from a different species. Antibodies tagging an antigen are then bound by fluorophore-conjugated secondary antibodies. Fluorophore-conjugated secondary antibodies should be selected so that they are compatible with the wavelengths of laser excitation available in the confocal microscope. When visualizing multiple antigens, the excitation/emission spectra of the fluorophores should differ enough so that their signals can be discriminated by microscopic analysis. The stained specimen is then mounted on a slide for imaging. A mounting medium is used to prevent photobleaching and specimen dehydration. If desired, a mounting medium containing a nuclear counterstain (e.g. DAPI or Hoechst) can be used (4).
In the following protocol, mouse fibroblasts transfected to express CD1d (LCD1) were stained with antibodies recognizing CD1d and CD107a (LAMP-1). CD1d is a major histocompatibility complex 1 (MHC 1)-like receptor present on the surface of antigen presenting cells that presents lipid antigens. LAMP-1 (lysosomal associated membrane protein-1) is a transmembrane protein primarily present in lysosomal membranes. For proper antigen presentation, CD1d is trafficked through the low pH lysosomal compartment, so LAMP-1 is being used as a marker of the lysosomal compartment for this protocol. By probing the LCD1 cells with anti-CD1d and anti- LAMP-1 that were produced in different species, secondary antibodies with unique fluorophores can be used to determine the localization of each protein in the cell and whether CD1d is present in the LAMP-1 positive lysosomal compartments.

Confocal Fluorescence Microscopy and Localization of Proteins

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.
The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.
In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Rag&#947;c mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10504/10504fig1.jpg" /> 
Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Rag&#947;c mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. <a href="https://www.jove.com/files/ftp_upload/10504/10504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Adoptive Cell Transfer Technique

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
One of the main functions of the cells of the immune system is to remove target cells that have been infected with viruses or have undergone transformation into a tumor cell. In vitro assays for measuring the cytotoxic capacity of immune cells have been a staple in laboratories for many years. These assays have been used to determine the ability of T cells, NK cells, or any other immune cell to kill target cells in an antigen-specific or -nonspecific manner. Death ligands (e.g., Fas ligand or TRAIL), cytokines (e.g., IFNg or TNF), or cytotoxic granules (i.e., perforin/granzyme B) expressed by effector cells are some ways in which target cell death can be induced. With the explosion in tumor immunotherapy research in recent years, there is growing interest in finding agents to increase the cytotoxic activity of immune cells to improve patient outcomes. Conversely, some diseases are hallmarked by the overexuberant activity of immune cell cytotoxic activity, resulting in efforts to identify agents to temper these responses. Thus, having an assay in which the user can easily integrate any number of different effector cells, target cells, and/or response modifiers into the experimental design can serve as a valuable means of quickly assessing the cytotoxic capacity of effector cells and/or the responsiveness of the target cell.
These in vitro assays involve the mixing of different cell populations, as well as using a relatively low number of both effector and target cells. Thus, one necessity of the assay is to label the target cells in a manner that can easily be detected and quantitated, allowing the user to then determine the &#39;percent specific lysis&#39; mediated by the effector cells. Radioactivity - especially, chromium 51 (51Cr) in the form of Na251CrO4- is an inexpensive way to rapidly and nonspecifically label cellular proteins within the target cells (1). The short labeling and total assay times reduces the potential for significant changes in the number and/or phenotype of the target cells, which could influence the outcome of the assay. Upon the loss of membrane integrity of the target cells as a result of the cytotoxic activity of the effector cells, the 51Cr-labelled cellular proteins within the target cells are released into the culture supernatant, becoming available for quantitation. As with any assay examining the function of immune cells in vitro, there are a number of important considerations to consider improving the performance of the experiment. One of the most critical features is to use healthy effector (for maximal cytotoxic activity) and target (for maximal responsiveness and minimal spontaneous death/51Cr release) cells. Effector and target cell contact is required (leading to the common use of round-bottom 96-well plates to encourage cell-cell contact) (2). Finally, data analysis is dependent on the inclusion of positive and negative control target cell populations.
The following protocol will outline the steps for performing a standard 51Cr release assay to measure the cytotoxic ability of a population of effector cells, though a nonradioactive version using Europium has recently been developed. 51Cr is a powerful &#947;-radiation emitter. Consequently, the use of this assay requires proper radiation safety training, dedicated laboratory space, a gamma counter, and disposal of radioactive samples.
The general sequence of events in this assay are: 1) prepare 51Cr-labeled targets; 2) prepare effector cells and add to plate while target cells are labeling; 3) add labeled targets to plate; 4) incubate plate; 5) harvest supernatants; and 6) analyze data after running samples on counter. Samples are commonly prepared in triplicate, and then averaged to account for any subtle pipetting differences.
Proper PPE is important for this assay. Specifically, the user should wear a lab coat and gloves. Safety glasses may be required based on the laboratory or institution. There should be ample lead shielding for safe storage and use of the 51Cr during all steps. Finally, there should be dedicated lab space and equipment set aside for using 51Cr, including all the proper signage to indicate where samples with 51Cr are being kept and a Geiger counter equipped with gamma probe to survey the space for possible contamination.
In this lab exercise, we will determine the ability human peripheral blood mononuclear cells (PBMCs), (CpG stimulated vs. unstimulated) to kill melanoma cells, using human melanoma cell line WM793 as model and the chromium release assay.

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