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.
However, when using magnetic levitation several points are worth mentioning. Firstly, the microscope used for imaging must be dedicated to this method, as the setup is time consuming, and requires the microscope to be partially disassembled, positioned perfectly horizontally with the optics precisely aligned with the magnets and the capillary tube. Secondly, the lab jack and the translational stages used to adjust the movements of the capillary glass and the focus require exact positioning and free movement on each of the three axes. Likely the most critical alignment of the entire setup, is mounting the magnetic levitation device in the translational stages such that the top and bottom magnets are perfectly aligned with the gravity vector as well as perfectly horizontal. Any deviation from these requirements&#160;would create an angle between the magnetic force and gravity which would push the cells toward either sides of the capillary tube, disrupting the levitation process, and making the results unreliable.
The optimal concentration of the gadolinium solution used for levitating cells needs to be adjusted for the cell type and the goal of the experiment. Changes in the concentration of the gadolinium solution will significantly alter the levitation height of the cells being analyzed, and therefore needs to be kept constant from one set of experiments to another. If the concentration is too low, depending on the density of the target cells, they may not levitate at all, while if it is too high, will limit the range of detectable changes that can be accurately quantified.
Levitating structures will create stable bands if the only forces acting on them are gravity and magnetic repulsion. The presence of even a millimeter-size bubble in the capillary tube will create small circular currents at the air-liquid interface, which will disturb the levitating cells, making any analysis virtually impossible. When loading a sample into a capillary tube, and then after sealing, one must make sure that no air is present, by examining the capillary under a stereomicroscope or low power (4x) objective.
Levitation of cells at a set height depends on their magnetic signature staying the same over time. If the paramagnetic gadolinium ions from the levitation media enters the cells, either through pinocytosis or increased membrane permeability, such as during apoptosis, the increased cell density measured based on the density reference beads will be erroneous. Fortunately, most of circulating cells have poor pinocytic capabilities26, making this method a suitable approach for studying cells over long periods of time.

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><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&quot;) thickness</td><td>K&amp;amp;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 (Gd&lt;sup&gt;3&lt;/sup&gt;+); 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&amp;prime;-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&amp;amp;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 reached by each cell type can easily be modified by changing the concentration of the paramagnetic solution. Figure 2 illustrates the separation of the different circulating blood cells by using various concentrations of gadolinium. The two bead types with different densities (1.05 and 1.2 g/mL) were used to provide density levitation heights and size references. As the levitation height of a given cell type depends on its intrinsic density, magnetic levitation provides a direct means of isolating cells of interest without any significant manipulation24. The main purpose of this protocol was to demonstrate the ability of magnetic levitation to detect the presence of membrane bound antigens, in this case the Rh factor, on circulating red blood cells. For this experiment, low density beads were coated with either IgG control antibody or anti-RhD. Samples were then prepared by spiking blood from an Rh(-) donor with blood from an Rh(+) at a ratio of 1:1000. Anti-RhD(+) coated beads or the control IgG coated beads were added to the blood sample and incubated for 10 minutes. Figure 3A shows the IgG control sample, which did not generate any bead-red blood cell complexes. Next, the identity of the red blood cells captured by the Rh(+)-positive beads was verified by pre-staining the Rh(+) cells with a fluorescent plasma membrane stain, and then imaged using fluorescence microscopy (Figure 3B). A positive detection event is shown in Figure 3C. The binding of the anti-Rh-bead to the Rh(+) cells creates a bead-cell complex with a density in between that of the beads and the cell, therefore levitating at a height situated in between the unbound capture beads and negative RBCs. A close up of the bead-cell complexes was imaged under fluorescent light to confirm the presence of the Rh(+) cells labeled with a fluorescent plasma membrane stain. (Figure 3D). Figure 3E shows bead-bead complexes forming between high- and low- density beads coated with antibodies for CR1 and CD47, which indicate the presence of extracellular vesicles. A schematic of the bead-cell complex is shown in Figure 3F.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62550/62550fig01.jpg" /> 
Figure 1. Principles of Magnetic Levitation: (A) Schematics of magnetic field. (B) A research grade microscope tipped on its side to allow side-imaging the targets levitating in the capillary tube. (C) Angled view of the magnetic levitation apparatus under the microscope objective. (D) Magnetic levitation apparatus frontal view. (E) Schematics of the magnetic levitation apparatus with a capillary tube mounted between two magnets, front and side view. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62550/62550fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62550/62550fig02.jpg" /> 
Figure 2. Demonstration of Cell-Specific Density Equilibrium: (A) Low- and high-density beads (1.05 and 1.2 g/mL) alone at 60 mM Gd3+ (viewed on a 10x objective). (B) PMNs levitating above red blood cells at 21 mM Gd3+, with out of focus platelets circulating (viewed on a 10x objective).&#160;(C) Whole blood levitating at 60 mM Gd3+, which is a concentration that focuses on RBCs (viewed on a 10x objective). (D) This figure has been modified from [Tasoglu, S. et al. Levitational Image Cytometry with Temporal Resolution. Advanced Materials.&#160;27 (26), 3901-3908, doi:10.1002/adma.201405660 (2015).]&#160;Density separation of RBCs (red), PMNs (green) and lymphocytes (blue) at 40 mM Gd3+. Each cell population levitated at a height based on their intrinsic densities (viewed on a 10x objective). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62550/62550fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62550/62550fig03.jpg" /> 
Figure 3. Detection of Rh Factor in a Blood Sample: (A) Rh(-) blood spiked with Rh(+) blood and IgG control beads. No bead-cell complexes are formed (viewed on a 10x objective). (B) IgG control sample under fluorescent light highlighting the Rh(+) cells (viewed on a 10x objective). (C) Rh(-) blood spiked with Rh(+) blood and anti-RhD coated beads, showing the formation of bead-cell complexes (viewed on a 10x objective).(D) A close-up view of a bead-cell complex under fluorescent light (viewed on a 10x objective).(E) Anti-CR1 and anti-CD47 coated beads forming complexes, indicating the presence of RBC derived extracellular vesicles (viewed on a 10x objective). (F) Diagram depicting the bead-cell complex. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62550/62550fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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2.7K 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

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Engineering

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Bioengineering

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 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Immunology and Infection

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

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.bdigitaluss.remotexs.co/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 ...

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Immunology

Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

Source: Thompson, L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62550">Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates the detection of the Rhesus (Rh) factor in blood samples using a magnetic levitation instrument. The magnetic field aligns and levitates beads and blood cells based on density. Treating the samples with anti-Rh antibodies results in intermediate-density bead-cell complexes&#39; formation, allowing for the differentiation of Rh-positive and Rh-negative red blood cells.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunodiagnostics

Cellular Immunodiagnostics

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...