1. Gt anti-Ms IgG Magnetic Particles, 1% w/v, Cat. # MM-40-10 or MMXA-40-10, ~2e8 particles/mL
2. Appropriate monoclonal antibody (anti-CD3, CD4 or CD8 etc.)
3. Appropriate FITC conjugates
4. Dulbecco PBS
5. Fetal bovine serum
7. Paraformaldehyde fixative
8. FlexiMag Separator, Jr., Cat. # FMJ-1000
SEPARATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD
Collect peripheral blood by venipuncture of the antecubital vein. Draw blood into heparin Vacutainer tubes, transfer to 50 mL polypropylene centrifuge tubes and dilute with an equal volume of calcium and magnesium-free Dulbecco’s phosphate buffered saline.
Layer 10 to 20 mL aliquotes of diluted blood onto an equal volume of Histopaque in 50 mL tubes. Centrifuge for 30 minutes at ambient temperature using a centrifugation force of 400g at the blood/Histopaque interface. Aspirate the lymphocyte band into a 15 mL centrifuge tube and bring the volume to 14 mL with Dulbecco PBS containing 2% v/v heat-inactivated fetal bovine serum, or 5% plasma protein fraction. Pellet the cells by centrifugation at 4°C for 7 minutes at 450g, wash with 14 mL of PBS, and recentrifuge. Resuspend the final cell pellet in 1-2 mL buffer, and a viable cell count performed. Only cell preparations with a viability of >95% should be used.
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