Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells
Transcrição
Before beginning the Vδ1 T cell isolation, use a 1 x 106 aliquot of the PBMC to assess the frequency of Vδ1CD4-positive T cells in the donor sample by flow cytometry. Then, spin down the rest of the lymphocytes, and re-suspend the pellet in 60 microliters of MACS buffer per 1 x 107 cells.
Next, block the cells in 20 microliters of Fc-blocking reagent per 1 x 107 cells, followed by the addition of 10 microliters of FITC-conjugated anti-human Vδ1 antibody per 1 x 107 cells for 12 minutes at 4 degrees Celsius in the dark. At the end of the incubation, wash the cells in 14 milliliters of MACS buffer.
Resuspend the pellet in 80 microliters of MACS buffer per 1 x 107 cells, and add 20 microliters of anti-FITC microbeads per 1 x 107 cells with mixing. After 15 minutes in the dark at 4 degrees Celsius, wash the cells in 10 milliliters of fresh MACS buffer.
During the centrifugation, equilibrate a pre-cooled magnetic column with 500 microliters of MACS buffer. Then, resuspend the cells in 500 microliters of MACS buffer, and add them carefully to the top of the column.
Rinse the column with three 500-microliter MACS buffer washes. Then, remove the column from the magnet, and use a plunger to quickly flush the Vδ1-positive cells with 1 milliliter of MACS buffer into a 15-milliliter conical tube.
After running the cells through a second column, vortex 25 microliters of CD4 beads for 30 seconds. Then, transfer the beads into a 1.5-milliliter reaction tube, containing 1 milliliter of MACS buffer, and place the tube on a magnet.
After 1 minute, use a 200-microliter pipette to carefully aspirate the supernatant, and resuspend the beads in 25 microliters of MACS buffer. Next, spin down the isolated Vδ1-positive cells and resuspend the pellet in 500 microliters of MACS buffer. Mix the cells vigorously with the beads. Then, after 20 minutes at 4 degrees Celsius, with constant tilting, place the tube back onto the magnet.
To avoid losing any sample volume and to minimize the presence of contaminating CD4-negative cells, it is essential to pipette any remaining buffer from the inside of the lid back into the vial.
After two minutes, use a 200-microliter pipette to transfer the CD4-negative Vδ1-positive cell-containing supernatant into a fresh tube. Then, remove the CD4-positive cells from the magnet, and resuspend them in 500 microliters of MACS buffer.
After removing the CD4-negative cells two more times, as just demonstrated, incubate the CD4-positive cells in 100 microliters of culture medium, and 10 microliters of bead-detaching solution at room temperature for 45 minutes with constant tilting. At the end of the incubation, return the cells to the magnet for 1 minute, and use a 200-microliter pipette to transfer the bead-free Vδ1-positive CD4-positive cell-containing supernatant into a new tube.
After removing any remaining beads two more times, as just demonstrated, spin down the cells. Then, resuspend the Vδ1-positive CD4-positive cells in fresh, pre-warmed medium for counting.
