I want to deplete (negative depletion) all T-cells and NK cells from human buffy coat PBMC using three antibodies: CD3-PE, CD16-PE and CD56-PE and anti-PE magnetic beads. Can I do that by mixing the antibodies and following the standard protocol? How to calculate how much antibodies and beads do I need? How to calculate the yield of total cells I would get in the flow-through? I'm assuming around 30-40%?
If anyone has ever had a similar problem I would really appreciate it if you shared your experience.
Yes, you can mix all the antibodies for MACS, that's resembling the original antibody cocktail of the separation kit. But to optimize or calculate the amount of antibodies, I would suggest to perform a preliminary test, try flow cytometry using the same antibodies, for example, 1e6 PBMC with 0.5 ug of each antibody and observe the positive percentage to predict the larger scale. Of note, antibodies need to be in excess to minimize the off-labeled cell contamination.
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A common starting point is around 1-5 µg of antibody per million cells.
Just some advice from experience- Don't use PE labeled antibodies for depletion. When I did this I consistently saw incomplete depletion despite titration of antibody and bead concentrations. Our miltenyi rep recommended using streptavidin (best) or APC-conjugated antibodies for depletion since there are more bead-binding sites per molecule (4-7 biotin molecules/antibody, and APC is dimeric with 2 sites). PE labeling is better for positive selection. Biotinylated antibodies are also generally much cheaper.
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