Name: quantification of cellular densities and antigenic properties using magnetic levitation
Uploaded: 2021-05-17T15:00:00
Description: Watch this Scientific Journal Video about Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation at JoVE.com

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 thi...

<ol>
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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 ...

This technique allows separation of various cell types and cells undergoing various processes based solely on density with little to no manipulation in a short period of time. The main advantages of this method are that it as minimally invasive, requires small volumes, provides fast results, does not depend on classic readouts, and specialized personnel is not required. This technique can be applied for detecting multiple diseases, for example anemia and sepsis.To begin use the one-click lancing device to prick the finger of the rhesus factor positive blood donor, and collect 10 microliters of blood in one milliliter of DPBS. Add one microliter of fluorescent dye in one milliliter suspension of the rhesus factor positive cells to fluorescently stained the plasma membrane and incubate at 37 degrees Celsius for 15 minutes. Pellet the cells by spinning at 5, 600 times G for 15 seconds and wash the pellet three times using one milliliter of DPBS.Resuspend the pellet in one milliliter of HBSS with calcium and magnesium. Use the one-click lancing device to prick the finger of the rhesus factor negative blood donor and collect two microliters of blood. To prepare the experimental tube containing only beads, add 174 microliters of HBSS with calcium and magnesium, one microliter of IgG control beads, one microliter of heavy beads with a 1.2 gram per milliliter density, and 24 microliters of 500 millimolar gadolinium.To prepare the experimental control tube containing IgG, add 172 microliters of HBSS with calcium and magnesium, one microliter of IgG control beads, one microliter of heavy beads, one microliter of rhesus factor negative blood, one microliter of stained rhesus factor positive blood suspension, and 24 microliters of 500 millimolar gadolinium. To prepare the sample experimental tube, add 172 microliters of HBSS with calcium and magnesium, one microliter of anti-RhD coated beads, one microliter of heavy beads, one microliter of rhesus factor negative blood, one microliter of stained rhesus factor positive blood suspension, and 24 microliters of 500 millimolar gadolinium. Set up the magnetic levitation device.And load 50 microliters of the sample into a capillary tube until the tube is filled. Seal the ends of the capillary tube with a capillary sealant ensuring that there are no bubbles. Place the capillary tube in the capillary holder between the top and bottom magnets, adjust the stage and focus for optimal viewing.Wait approximately five to 20 minutes for the cells and beads to settle at their magnetic equilibrium position. Using this protocol, the blood cells were separated depending upon the levitation height using the magnetic levitation device. Low and high density beads were used to provide density levitation heights and size references.The polymorphonuclear cells were levitating above the red blood cells at 21 millimolar gadolinium ion concentration. Old blood cells were levitating at 60 millimolar gadolinium ion concentration. The red blood cells, polymorphonuclear cells, and lymphocytes were separated at 40 millimolar gadolinium ion concentration based on their intrinsic densities.The rhesus factor antigen was detected on the red blood cells using IgG and anti-RhD antibody coated beads. The bead red cell complexes were not generated in the IgG control sample, but the fluorescently stained red blood cells captured by the rhesus factor positive beads were detected. The binding of the anti-RhB to the rhesus factor positive cells created a bead cell complex in the center at intermittent density of the unbound beads and the negative red blood cells.Further, the complex formation was confirmed by observing the emitted fluorescence. The B bead complexes formed between the high and low density beads coated with the antibodies for CR1 and CD47 indicate the presence of red blood cell derived extracellular vesicles. Capping the capillary without introducing bubbles is the most challenging step and requires practice.This method could provide insight for hematology, especially focused on red blood cell diseases.

Research

JoVE Journal

Biology

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