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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

Published: May 17th, 2021

DOI:

10.3791/62550

1Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center

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 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.

Magnetic levitation is a technique developed to separate, analyze, and identify cell 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<....

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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.......

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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 .......

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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 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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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.

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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

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