Source: Thompson, L. et al., Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation. J. Vis. Exp. (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' 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 welfare and have been reviewed by the local institutional review board.
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 1×1 mm squared glass capillary tube. The device was 3-D printed using an AutoCAD design, which is available upon request.
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 Rhesus, Rh(+) detection, but both high- and low-density beads are coated for the detection of extracellular vesicles.
3. Collection and Preparation of Blood for Rh(+) Detection
4. Analyzing Cells on the Magnetic Levitation Device
The authors have nothing to disclose.
2-(N-Morpholino)ethanesulfonic acid hydrate | Sigma Aldrich | M-2933 | (MES); component of activation buffer |
50×2.5×1 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 |
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 |