Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 setsĀ 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 cellĀ 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<....

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 thi.......

Representative 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 Kƶhler 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 hereā€Æallows 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.

.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
2-(N-Morpholino)ethanesulfonic acid hydrateSigma AldrichM-2933(MES); component of activation buffer
50x2.5x1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197") thicknessK&J MagneticsCustomMagnets used for the magnetic levitation device
Capillary Tube Sealant (Critoseal)Leica Microsystems267620Used to cap the ends of the capillary tubes
Centrifuge tube filters (Corning Costar Spin-X)Sigma AldrichCLS8163Used to wash beads
Compact Lab JackThorlabsLJ750Used for adjusting the magnetic levitation device
DPBS, no calcium, no magnesiumGibco14190-144Solution for bead suspensions
EthanolamineSigma AldrichE9508-100MLUsed during a wash step for beads
Fluorescent Plasma Membrane Stain (CellMask Green)InvitrogenC37608Used to stain Rh+ cells
Gadoteridol InjectionProHanceNDC 0270-1111-03Gadolinium (Gd3+); magnetic solution used to suspend cells
HBSS++Gibco14025-092Solution for sample preparation
Human C5b,6 complexComplement Technology, IncA122Used to generate RBC Evs
Human C7 proteinComplement Technology, IncA124Used to generate RBC Evs
Human C8 proteinComplement Technology, IncA125Used to generate RBC Evs
Human C9 proteinComplement Technology, IncA126Used to generate RBC Evs
Mini Series Post CollarThorlabsMSR2Used to secure magnetic levitation device to lab jacks
N-(3-Dimethylaminopropyl)-Nā€²-ethylcarbodiimide hydrochlorideSigma AldrichE1769-10G(EDC); used in antibody coupling reaction
Normal Rabbit IgG ControlR&D SystemsAB-105-CUsed to coat beads as a control condition
Phosphate Buffered Saline (10X Solution, pH 7.4)Boston BioproductsBM-220Component of coupling buffer, used for washing steps
Polysorbate 20 (Tween 20)Sigma AldrichP7949-500MLComponent of activation buffer
Polystyrene Carboxyl PolymerBangs LaboratoriesPC06004Top density beads (1.05 g/mL), used for antibody coupling
Rabbit RhD Polyclonal AntibodyInvitrogenPA5-112694Used to coat beads for the dectection of Rh factor in red blood cells
Research Grade MicroscopeOlympusProvis AX-70Microscoped used to mount magnetic levitation device and view levitating cells
Rubber Dampening FeetThorlabsRDF1Used to support the breadboard table
Square Boro TubingVitroTubes8100-050Capillary tube used for loading sample into Maglev
Sulfo-NHSThermoscientific24510Used in antibody coupling reaction
Translational StageThorlabsPT1Used for focusing and for scanning capillary tube

References

  1. Tasoglu, S., et al. Levitational Image Cytometry with Temporal Resolution. Advanced Materials. 27 (26), 3901-3908 (2015).
  2. Durmus, N. G., et al. Magnetic levitation of single cells. Proceedings of the National Academy of Sciences....

Explore More Articles

Magnetic LevitationCell DensityAntigenic PropertiesMinimally InvasiveRhesus FactorFluorescent DyeDPBSHBSSGadoliniumIgG Control BeadsAnti RhD Coated BeadsCapillary TubeCell SeparationCell DetectionAnemiaSepsis

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright Ā© 2024 MyJoVE Corporation. All rights reserved