This study demonstrates a method for isolating Rhesus factor-positive red blood cells (RBCs) using a magnetic levitation device. By utilizing paramagnetic solutions and antibody-coated beads, the technique allows for the differentiation of blood samples based on density.
Take a paramagnetic gadolinium solution responsive to external magnetic fields.
Introduce fluorescently labeled Rhesus or Rh factor-positive and unlabeled Rh factor-negative blood samples.
Add anti-RhD antibody-coated low-density polymer beads and uncoated high-density beads.
Anti-RhD antibodies bind to RhD-antigens on Rh-positive RBCs, forming bead-RBC complexes.
Now, load the sample into a capillary tube and seal it. Place the tube between magnets in the magnetic levitation device.
In the magnetic field generated by like-poles-facing magnets, the paramagnetic solution exerts magnetic and buoyant forces on sample components, causing them to levitate at heights proportional to their densities.
Low-density and high-density beads levitate at the top and bottom of the capillary, respectively, establishing density height references.
Blood cells, being diamagnetic, are repelled by a strong magnetic field near the magnets and levitate at an intermediate position.
The bead-RBC complexes levitate between low-density beads and RBCs, with fluorescent cells in this layer confirming Rh-positive RBC presence.
To begin, use the one-click lancet device to prick the finger of the Rhesus factor-positive blood donor, and collect 10 microliters of blood in 1 milliliter of DPBS. Add 1 microliter of fluorescent dye in 1-milliliter suspension of the Rhesus factor-positive cells to fluorescently stain the plasma membrane, and incubate at 37 degrees Celsius for 15 minutes. Pellet the cells by spinning at 5,600 times g for 15 seconds, and wash the pellet three times, using 1 milliliter of DPBS. Resuspend the pellet in 1 milliliter of HBSS with calcium and magnesium. Use the one-click lancing device to prick the finger of the Rhesus factor-negative blood donor, and collect two microliters of blood.
To prepare the experimental tube containing only beads, add 174 microliters of HBSS with calcium and magnesium, 1 microliter of IgG control beads, 1 microliter of heavy beads, with a 1.2 gram per milliliter density, and 24 microliters of 500 millimolar gadolinium.
To prepare the experimental control tube containing IgG, add 172 microliters of HBSS with calcium and magnesium, 1 microliter of IgG control beads, 1 microliter of heavy beads, 1 microliter of Rhesus factor-negative blood, 1 microliter of stained rhesus factor-positive blood suspension, and 24 microliters of 500 millimolar gadolinium.
To prepare the sample experimental tube, add 172 microliters of HBSS with calcium and magnesium 1 microliter of anti-RhD coated beads, 1 microliter of heavy beads, 1 microliter of rhesus factor-negative blood, 1 microliter of stained rhesus factor-positive blood suspension, and 24 microliters of 500 millimolar gadolinium. Set up the magnetic levitation device, and load 50 microliters of the sample into a capillary tube until the tube is filled.
Seal the ends of the capillary tube with a capillary sealant, ensuring that there are no bubbles. Place the capillary tube in the capillary holder between the top and bottom magnets. Adjust the stage, and focus for optimal viewing. Wait approximately 5 to 20 minutes for the cells and beads to settle at their magnetic equilibrium position.
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