We have been using in our lab MACS for cell separation but the retrieval has been low. If we add 20 million PBMC, we only retrieve about 2-3 million.
Hi Juan,
your recovery is indeed at the low side. As more than half of the PBMCs usually consist of CD3+ cells (although this is species dependent), you should be able to purify at least 50% of your cells as CD3+. I'm guessing that either the concentration of your aCD3 mAb or the incubation time is suboptimal. If you could provide more details on your protocol, I (or others) should be able to pinpoint your problem.
Thanks Bert,
We add 1uL CD3 microbeads for every million PBMCs. We usually try to isolate CD3+ from 20million PBMC, so we add 20uL CD3 microbeads we add 80 uL of Buffer (PBS 1x + 0.5% BSA + 2mM EDTA). We incubate them for 20 minutes in darkness at 4C. We wash them with 3 mL of buffer and centrifuge them at 200 g for 10 minutes. Then we resuspend them with 3 mL of Buffer, and we add it to the column, we collect the negative pass through. We change the tube, and remove the column from the magnet, and we collect the positive cells.
The aCD3 bead concentration seems okay. I would increase the incubation time to 30 min. You might also lose some cells in your centrifugation step. I would increase till 300-400 g instead of 200 g. Maybe you could also stain your negative fraction for CD3 to determine the efficiency of your MACS.
Good luck!
Thanks for your help, I have stained the negative fraction and it is around 50%, which is high.
MACS islation is available with diffrerent magnetic columns, what about trying with another longer column. Perhaps because of your hich number of cells you are saturating your column.
Hi Juan, You might would like to try the pluriBead system. You can use directly in whole blood, buffy coat or PBMC. You can adjust the numbers of CD3 cells that you woul like to isolate with the numbers of beads. Independend of the numbers of total cells. You do not need magnets, the cells are bead free at the end. please check here: http://pluriselect.com/product-details/product/cd3-s-pluribeads-anti-hu.html. In case you have questions please do not hesitate to ask. Best wishes Michael
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