Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

Summary

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.

Abstract

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 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.

Introduction

Magnetic levitation is a technique developed to separate, analyze, and identify cell types^1,2,3, proteins^4,5 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 status^{7,8,9,10,11,12,13,14}. 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 differentiation^15,16, cell cycle progression^9,17,18,19, apoptosis^20,21,22,23, and malignant transformation^24,25,26. 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 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'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.

Protocol

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 mm squared glass capillary tube. The device was 3-D printed using an AutoCAD design, which is available upon request.

1. Lay microscope on its side, perfectly horizontal.
   ​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 Köhler illumination requirements.
2. Support and level the stand on a breadboard table using 2 or 3 lab jacks.
3. To limit vibrations, support the breadboard table with rubber dampening feet.
4. 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.
5. Attach the magnetic levitation device to the lab jack using two mini-series optical posts.

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.

1. 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).
2. Add 12 µL of freshly made 1.5 M EDC (MW 191.7, 0.28755 g in 1 mL) and 12 µL of 0.3 M Sulfo-NHS (MW 217.14, 0.0651 g in 1 mL) in ice-cold water.
3. Place tubes on an orbital shaker and shake vigorously for 1 h at room temperature to activate the carboxyl groups on beads.
4. Stop the activation after 1 h by adding 0.5 mL of Coupling Buffer (10x PBS, or 0.1 M phosphate pH 7-9).
5. Pellet the beads by centrifuging at 20,000 x g for 10 min, or, if the beads cannot be pelleted, use a 0.45 µm centrifuge tube filter. Aspirate the supernatant or flow-through.
6. Wash the beads with 1 mL of 10x PBS 3 times as in step 2.5.
7. Calculate 25 µg 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.
8. Place tubes on a tube rocker set on a low speed. Roll tubes gently at room temperature overnight for coupling.
9. Repeat step 2.5 and wash the beads twice with 1 mL of 10x PBS.
10. 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.
11. Repeat step 2.5 and wash the beads once in 1 mL of DPBS. The beads can be stored in 200 µL of DPBS at 4 °C until needed.
    ​NOTE: The protocol can be paused here.

3. Collection and Preparation of Blood for Rh(+) Detection

1. Using a one-click lancing device, prick the finger of an Rh(+) donor and collect 10 µL of blood into 1 mL of DPBS.
2. Stain the Rh+ cells with a fluorescent plasma membrane stain. Optionally, add 1 µL of fluorescent dye to the 1 mL suspension of Rh+ cells (1:1000 dilution).
3. Incubate the cells with the fluorescent dye at 37 °C for 15 min.
4. 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++).
5. Using the one-click lancing device, prick the finger of an Rh(-) donor and collect 2 µL of blood.
   NOTE: If preparing more than 2 conditions, collect enough blood to add 1 µL of Rh(-) blood to each tube.
6. Prepare the necessary experimental tubes: Beads alone, IgG control, sample.
   1. Beads alone: add 174 µL of HBSS++, 1 µL of IgG control beads, 1 µL of high-density beads (1.2 g/mL), and 24 µL of 500 mM Gd³⁺ (60 mM).
   2. IgG control: add 172 µL of HBSS++, 1 µL of IgG control beads, 1 µL of high-density beads, 1 µL of Rh- blood, 1 µL of stained Rh+ blood suspension, and 24 µL of 500 mM Gd³⁺.
   3. Sample tube add 172 µL of HBSS++, 1 µL of anti-RhD coated beads, 1 µL of high-density beads, 1 µL of Rh- blood, 1 µL of stained Rh(+) blood suspension, and 24 µL of 500 mM Gd³⁺.
      ​NOTE: High density beads are added to Rh samples for reference.

4. Isolation of PMNs for Cell Separation Demonstration

1. Isolation of Neutrophils
   1. 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.
   2. Wait for 50 min for blood to sediment.
   3. 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.
   4. 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.
   5. Transfer the neutrophils to a new 50 mL tube.
   6. 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).
   7. Centrifuge the suspension for 10 min at 3,000 x g.
   8. Remove the supernatant and resuspend the neutrophils to the desired concentration.
2. Isolation of Lymphocytes
   1. Plate the PBMCs in RPMI with 5% heat inactivated serum in 6-well culture plates.
   2. Incubate the plates for 1 h at 37 °C. Monocytes will adhere to the plate, lymphocytes will be freely floating.
   3. Remove the buffer containing the lymphocytes and wash it twice with RPMI.
   4. Resuspend the lymphocytes at the desired concentration.
3. Label RBCs, PMN, and Lymphocytes.
   1. Label each cell type with a different fluorescent dye. Make sure each dye fluoresces in a different channel. Follow the manufacturer's instructions for each of the chosen dyes.

5. Generation of RBC Extracellular Vesicles via the Complement Activation

1. Obtain whole blood through venipuncture using EDTA tubes.
2. 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.
3. Make aliquots of 100 μL packed RBCs in 1.5 mL tubes and make up the volume to 1 mL by adding HBSS++.
4. Add C5b,6 to a 0.18 μg/mL final concentration in HBSS++, and then vortex.
5. Put on a slow shaker at room temperature for 15 min.
6. Add C7 protein to a final concentration of 0.2 μg/mL. Mix by inverting the tube gently a few times. Do not vortex from this point forward.
7. Place the tube on a tube shaker set at a low speed for 5 min at room temperature.
8. Add C8 protein to a final concentration of 0.2 μg/mL, and C9 protein to 0.45 μg/mL. Mix by inverting the tubes gently a few times.
9. Incubate at 37 °C for 30 min.
10. Centrifuge the tube at 10,000 x g for 10 min.
11. Collect the EV-containing supernatant in a new tube(s). Do not tremove the supernatant too close to the cell pellet.

6. Analyzing Cells on the Magnetic Levitation Device

1. Perform instrument startup according to manufacturer instructions.
2. Load 50 µL 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.
3. 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.

Results

Magnetic levitation focuses objects of different densities at different levitation heights depending on the object'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 Köhler illumination, by using a microscope turned on its side (Figure 1). The final levitation height ...

Discussion

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 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.

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Materials

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