The authors would like to thank Dr. Getulio Pereira for his help with extracellular vesicle work.
This work was supported by the following National Institute of Health grants to ICG: RO1CA218500, UG3HL147353, and UG3TR002881.

The experimental protocol used in this study was approved by the Beth Israel Deaconess Medical Center Institutional Review Board (IRB).
1. Instrument setup
NOTE: Imaging levitating cells requires two rare earth neodymium magnets magnetized on the z-axis to be placed with the same pole facing each other to generate a magnetic field. The distance between the magnets can be customized depending on the intensity of the magnetic field and the density of the targets. In this case the magnets are separated by a 1mm space sufficient for insertion of a 50 mm long 1x1&#160;mm squared glass capillary tube. The device was 3-D printed using an AutoCAD design, which is available upon request.
<ol>
	<li>Lay microscope on its side, perfectly horizontal. 
	&#8203;NOTE: A microscope placed in a standard upright position is not directly suitable for imaging levitating objects due to the positions of the magnets with respect to condenser and objective. This limitation can be bypassed by laying the microscope on its side, perfectly horizontal allowing the condenser to focus the light into the capillary, and the objective lens to image the cell levitating in between the magnets, while maintaining K&#246;hler illumination requirements.</li>
	<li>Support and level the stand on a breadboard table using 2 or 3 lab jacks.</li>
	<li>To limit vibrations, support the breadboard table with rubber dampening feet.</li>
	<li>Remove the stage and replace it with a compact lab jack for adjusting the height of the levitation device (y-axis), and two single-axis translational stages; one for adjusting the focus (z-axis), and the second one for scanning the capillary tube.</li>
	<li>Attach the magnetic levitation device to the lab jack using two mini-series optical posts.</li>
</ol>
2. Binding of Antibody to Carboxy-Microparticles/Beads (Modified from a protocol by PolyAn)
NOTE: Only low-density beads (1.05 g/mL) need to be coated for Rh(+) detection, but both high- and low-density beads are coated for the detection of extracellular vesicles.
<ol>
	<li>Take out 1 mg equivalent of bead suspension and add into 0.5 mL of Activation Buffer (50 mM MES (MW195.2, 9.72 mg in 1 mL)) pH 5.0 and 0.001% Polysorbate-20).</li>
	<li>Add 12 &#181;L of freshly made 1.5 M EDC (MW 191.7, 0.28755 g in 1 mL) and 12 &#181;L of 0.3 M Sulfo-NHS (MW 217.14, 0.0651 g in 1 mL) in ice-cold water.</li>
	<li>Place tubes on an orbital shaker and shake vigorously for 1 h at room temperature to activate the carboxyl groups on beads.</li>
	<li>Stop the activation after 1 h by adding 0.5 mL of Coupling Buffer (10x PBS, or 0.1 M phosphate pH 7-9).</li>
	<li>Pellet the beads by centrifuging at 20,000 x g for 10 min, or, if the beads cannot be pelleted, use a 0.45 &#181;m centrifuge tube filter. Aspirate the supernatant or flow-through.</li>
	<li>Wash the beads with 1 mL of 10x PBS 3 times as in step 2.5.</li>
	<li>Calculate 25 &#181;g of antibody per mg beads and mix desired antibody with activated beads to a 0.7-1.0 mg/mL final antibody concentration in 10X PBS.</li>
	<li>Place tubes on a tube rocker set on a low speed. Roll tubes gently at room temperature overnight for coupling.</li>
	<li>Repeat step 2.5 and wash the beads twice with 1 mL of 10x PBS.</li>
	<li>Wash with 0.5 mL of 1 M ethanolamine (98% stock = 16.2 M) in buffer pH 8.0 with 0.02% Polysorbate-20 for 1 h at room temperature while gently shaking.</li>
	<li>Repeat step 2.5 and wash the beads once in 1 mL of DPBS. The beads can be stored in 200 &#181;L of DPBS at 4 &#176;C until needed. 
	&#8203;NOTE: The protocol can be paused here.</li>
</ol>
3. Collection and Preparation of Blood for Rh(+) Detection
<ol>
	<li>Using a one-click lancing device, prick the finger of an Rh(+) donor and collect 10 &#181;L of blood into 1 mL of DPBS.</li>
	<li>Stain the Rh+ cells with a fluorescent plasma membrane stain. Optionally, add 1 &#181;L of fluorescent dye to the 1 mL suspension of Rh+ cells (1:1000 dilution).</li>
	<li>Incubate the cells with the fluorescent dye at 37 &#176;C for 15 min.</li>
	<li>Pellet the cells by spinning at 5,600 x g for 15 s and wash 3 times using 1 mL of DPBS. Resuspend in 1 mL of HBSS with calcium and magnesium (HBSS++).</li>
	<li>Using the one-click lancing device, prick the finger of an Rh(-) donor and collect 2 &#181;L of blood. 
	NOTE: If preparing more than 2 conditions, collect enough blood to add 1 &#181;L of Rh(-) blood to each tube.</li>
	<li>Prepare the necessary experimental tubes: Beads alone, IgG control, sample.
	<ol>
		<li>Beads alone: add 174 &#181;L of HBSS++, 1 &#181;L of IgG control beads, 1 &#181;L of high-density beads (1.2 g/mL), and 24 &#181;L of 500 mM Gd3+ (60 mM).</li>
		<li>IgG control: add 172 &#181;L of HBSS++, 1 &#181;L of IgG control beads, 1 &#181;L of high-density beads, 1 &#181;L of Rh- blood, 1 &#181;L of stained Rh+ blood suspension, and 24 &#181;L of 500 mM Gd3+.</li>
		<li>Sample tube add 172 &#181;L of HBSS++, 1 &#181;L of anti-RhD coated beads, 1 &#181;L of high-density beads, 1 &#181;L of Rh- blood, 1 &#181;L of stained Rh(+) blood suspension, and 24 &#181;L of 500 mM Gd3+. 
		&#8203;NOTE: High density beads are added to Rh samples for reference.</li>
	</ol>
	</li>
</ol>
4. Isolation of PMNs for Cell Separation Demonstration
<ol>
	<li>Isolation of Neutrophils
	<ol>
		<li>Draw 40 mL of venous blood into a 60 mL syringe containing 6 mL of sodium citrate/citric acid (0.15 M, pH 5.5) and 14 mL of 6% Dextran-70.</li>
		<li>Wait for 50 min for blood to sediment.</li>
		<li>Slowly layer the buffy coat cells on top of 20 mL of Ficoll-Paque by pushing the top 18 mL through the blood collection tubing into a 50 mL tube, avoiding contamination with sedimented RBCs. It is recommended to use a fresh blood collection set, to minimize the contamination with residual RBCs left over in the original blood collection tube.</li>
		<li>Pellet the buffy coat cells by centrifugation for 20 min at 3,000 x g. Neutrophils and contaminating RBCs will pellet at the bottom of the tube. PBMC will form a white layer on top of Ficoll-Paque.</li>
		<li>Transfer the neutrophils to a new 50 mL tube.</li>
		<li>Lyse any residual RBCs by incubating the neutrophils with 20 mL of 0.2% cold NaCl solution for 25 seconds, followed by an additional 20 mL of 1.6% NaCl. The final concertation of NaCl should be 0.9% (isotonic).</li>
		<li>Centrifuge the suspension for 10 min at 3,000 x g.</li>
		<li>Remove the supernatant and resuspend the neutrophils to the desired concentration.</li>
	</ol>
	</li>
	<li>Isolation of Lymphocytes
	<ol>
		<li>Plate the PBMCs in RPMI with 5% heat inactivated serum in 6-well culture plates.</li>
		<li>Incubate the plates for 1 h at 37 &#176;C. Monocytes will adhere to the plate, lymphocytes will be freely floating.</li>
		<li>Remove the buffer containing the lymphocytes and wash it twice with RPMI.</li>
		<li>Resuspend the lymphocytes at the desired concentration.</li>
	</ol>
	</li>
	<li>Label RBCs, PMN, and Lymphocytes.
	<ol>
		<li>Label each cell type with a different fluorescent dye. Make sure each dye fluoresces in a different channel. Follow the manufacturer&#39;s instructions for each of the chosen dyes.</li>
	</ol>
	</li>
</ol>
5. Generation of RBC Extracellular Vesicles via the Complement Activation
<ol>
	<li>Obtain whole blood through venipuncture using EDTA tubes.</li>
	<li>Pass blood through a white blood cell filter, and then centrifuge 3 times at 500 x g for 10 min each to isolate red blood cells. Use HBSS++ as washing buffer.</li>
	<li>Make aliquots of 100 &#956;L packed RBCs in 1.5 mL tubes and make up the volume to 1 mL by adding HBSS++.</li>
	<li>Add C5b,6 to a 0.18 &#956;g/mL final concentration in HBSS++, and then vortex.</li>
	<li>Put on a slow shaker at room temperature for 15 min.</li>
	<li>Add C7 protein to a final concentration of 0.2 &#956;g/mL. Mix by inverting the tube gently a few times. Do not vortex from this point forward.</li>
	<li>Place the tube on a tube shaker set at a low speed for 5 min at room temperature.</li>
	<li>Add C8 protein to a final concentration of 0.2 &#956;g/mL, and C9 protein to 0.45 &#956;g/mL. Mix by inverting the tubes gently a few times.</li>
	<li>Incubate at 37 &#176;C for 30 min.</li>
	<li>Centrifuge the tube at 10,000 x g for 10 min.</li>
	<li>Collect the EV-containing supernatant in a new tube(s). Do not tremove the supernatant too close&#160;to the cell pellet.</li>
</ol>
6. Analyzing Cells on the Magnetic Levitation Device
<ol>
	<li>Perform instrument startup according to manufacturer instructions.</li>
	<li>Load 50 &#181;L of sample into a capillary tube until the tube is filled. Seal the ends of the capillary tube with capillary sealant making sure there are no air bubbles present.</li>
	<li>Load the capillary tube into the holder between the top and bottom magnets. Adjust the stage and focus for optimal viewing. 
	NOTE: Cells/beads can take anywhere from 5-20 min to reach their magnetic equilibrium position based on their density and the concentration of Gd3+. The higher the concentration of Gd3+, the shorter the time.</li>
</ol>

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The authors have no conflicts to disclose.

Gradient centrifugation is currently the standard technique for isolating subcellular components based on their unique densities. This approach, however, requires the use of specialized gradient media as well as centrifuge equipment. The magnetic levitation approach presented here&#8239;allows detailed investigation of the morphological and functional properties of circulating cells, with minimum, if any manipulation of the cells, providing a near in vivo access to circulating cells.
...

Magnetic levitation is a technique developed to separate, analyze, and identify cell&#160;types1,2,3, proteins4,5 and opioids6 based solely on their specific density and paramagnetic properties. Cell density is a unique, intrinsic property of each cell type directly related to its metabolic rate and differentiation status7,8,9,10,11,12,13,14. Quantifying subtle and transient changes in cell density during steady state conditions, and during a variety of cell processes, could afford one an unmatched insight into cell physiology and pathophysiology. Changes in cell density are associated with cell differentiation15,16, cell cycle progression9,17,18,19, apoptosis20,21,22,23, and malignant transformation24,25,26. Therefore, quantification of specific changes in cell density, can be used to differentiate between cells of different types, as well as discriminate between same type of cells undergoing various activation processes. This enables experiments that target a particular cell sub-population, where dynamic changes in density serves as an indicator of altered cell metabolism27. As it has been established that a cell may alter its density in response to a changing environment7, it is imperative to measure the kinetics of the cell in relation to its density to understand it fully, which current methods may not provide12. Magnetic levitation on the other hand, allows for a dynamic evaluation of cells and their properties28.
Cells are diamagnetic meaning that they do not have a permanent magnetic dipole moment. However, when exposed to&#160;an external magnetic field, a weak magnetic dipole moment is generated in the cells, in the opposite direction of the applied field. Thus, if cells are suspended in a paramagnetic solution and exposed to a strong vertical magnetic field, they will levitate away from the magnetic source and stop to a height, which depends primarily on their individual density. Diamagnetic levitation of an object confined to the minimum of an inhomogeneous magnetic field is possible when the two following criteria are fulfilled: 1) the magnetic susceptibility of the particle must be smaller than that of the surrounding medium, and 2) the magnetic force must be strong enough to counterbalance the particle&#39;s buoyancy force. Both criteria can be fulfilled by suspending RBCs in a magnetic buffer and by creating strong magnetic field gradients with small, inexpensive, commercially available permanent magnets1. The equilibrium position of a magnetically trapped particle on an axis along the direction of gravity is determined by its density (relative to the density of the buffer), its magnetic susceptibility (relative to the magnetic susceptibility of the buffer), and the signature of the applied magnetic field. As the density and the magnetic properties of the solution are constant throughout the system, the intrinsic density properties of the cells will be the major factor determining the levitation height of the cells, with denser cells levitating lower compared to less dense cells. This approach uses a set of two density reference beads (1.05 and 1.2 g/mL) that allows us to use precise, ratiometric analysis for density measurements. Altering the concentration of the magnetic solution allows one to isolate different cellular populations, such as RBCs from WBCs, as the density of circulating cells is cell specific, removing the need for isolation protocols or other cell manipulation.
The majority of detection methods used in biology research rely on extrapolation of specific binding events into easy to quantify linear signals. These readout methods are often complex and involve specialized equipment and dedicated scientific personnel. An approach aimed at the detection of antigens found either on the plasma membrane of cells or extracellular vesicles or that are soluble in plasma, using either one or two antibody coated beads, is herein described. The beads must be of different densities from each other and from those of the interrogated targets. The presence of the target antigen in any given biofluid is translated into a specific, measurable change in the levitation height of an antigen-positive cell that is bound to a detection bead. In the case of soluble antigens or extracellular vesicles, they are bound to both capture and detection beads, forming a bead-bead complex rather than bead-cell complex. The change in levitation height depends on the new density of the bead-cell or bead-bead complexes. In addition to the change in the levitation height of the complexes, which indicates the presence of antigen in the biofluid, the number of complexes is also dependent on the amount of target, making magnetic levitation also a quantitative approach for antigen detection24.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid hydrate</td>
			<td>Sigma Aldrich</td>
			<td>M-2933</td>
			<td>(MES); component of activation buffer</td>
		</tr>
		<tr>
			<td>50x2.5x1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197&#34;) thickness</td>
			<td>K&#38;J Magnetics</td>
			<td>Custom</td>
			<td>Magnets used for the magnetic levitation device</td>
		</tr>
		<tr>
			<td>Capillary Tube Sealant (Critoseal)</td>
			<td>Leica Microsystems</td>
			<td>267620</td>
			<td>Used to cap the ends of the capillary tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tube filters (Corning Costar Spin-X)</td>
			<td>Sigma Aldrich</td>
			<td>CLS8163</td>
			<td>Used to wash beads</td>
		</tr>
		<tr>
			<td>Compact Lab Jack</td>
			<td>Thorlabs</td>
			<td>LJ750</td>
			<td>Used for adjusting the magnetic levitation device</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Solution for bead suspensions</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma Aldrich</td>
			<td>E9508-100ML</td>
			<td>Used during a wash step for beads</td>
		</tr>
		<tr>
			<td>Fluorescent Plasma Membrane Stain (CellMask Green)</td>
			<td>Invitrogen</td>
			<td>C37608</td>
			<td>Used to stain Rh+ cells</td>
		</tr>
		<tr>
			<td>Gadoteridol Injection</td>
			<td>ProHance</td>
			<td>NDC 0270-1111-03</td>
			<td>Gadolinium (Gd3+); magnetic solution used to suspend cells</td>
		</tr>
		<tr>
			<td>HBSS++</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>Solution for sample preparation</td>
		</tr>
		<tr>
			<td>Human C5b,6 complex</td>
			<td>Complement Technology, Inc</td>
			<td>A122</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C7 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A124</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C8 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A125</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Human C9 protein</td>
			<td>Complement Technology, Inc</td>
			<td>A126</td>
			<td>Used to generate RBC Evs</td>
		</tr>
		<tr>
			<td>Mini Series Post Collar</td>
			<td>Thorlabs</td>
			<td>MSR2</td>
			<td>Used to secure magnetic levitation device to lab jacks</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>E1769-10G</td>
			<td>(EDC); used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>AB-105-C</td>
			<td>Used to coat beads as a control condition</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X Solution, pH 7.4)</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td>Component of coupling buffer, used for washing steps</td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Sigma Aldrich</td>
			<td>P7949-500ML</td>
			<td>Component of activation buffer</td>
		</tr>
		<tr>
			<td>Polystyrene Carboxyl Polymer</td>
			<td>Bangs Laboratories</td>
			<td>PC06004</td>
			<td>Top density beads (1.05 g/mL), used for antibody coupling</td>
		</tr>
		<tr>
			<td>Rabbit RhD Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>PA5-112694</td>
			<td>Used to coat beads for the dectection of Rh factor in red blood cells</td>
		</tr>
		<tr>
			<td>Research Grade Microscope</td>
			<td>Olympus</td>
			<td>Provis AX-70</td>
			<td>Microscoped used to mount magnetic levitation device and view levitating cells</td>
		</tr>
		<tr>
			<td>Rubber Dampening Feet</td>
			<td>Thorlabs</td>
			<td>RDF1</td>
			<td>Used to support the breadboard table</td>
		</tr>
		<tr>
			<td>Square Boro Tubing</td>
			<td>VitroTubes</td>
			<td>8100-050</td>
			<td>Capillary tube used for loading sample into Maglev</td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Thermoscientific</td>
			<td>24510</td>
			<td>Used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Translational Stage</td>
			<td>Thorlabs</td>
			<td>PT1</td>
			<td>Used for focusing and for scanning capillary tube</td>
		</tr>
	</tbody>
</table>

quantification of cellular densities and antigenic properties using magnetic levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

Magnetic levitation focuses objects of different densities at different levitation heights depending on the object&#39;s density, its magnetic signature, the concentration of paramagnetic solution, and the strength of the magnetic field created by two strong, rare-earth magnets. As the two magnets are placed on top of each other, levitating samples can only be viewed, while maintaining K&#246;hler illumination, by using a microscope turned on its side (Figure 1). The final levitation height ...

Watch this Scientific Journal Video about Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation at JoVE.com

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

quantification-cellular-densities-antigenic-properties-using-magnetic

Research

JoVE Journal

Biology

2.6K Views.  Beth Israel Deaconess Medical Center, Harvard Medical School. This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

Video: Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...