Hello all,

I'm currently working with PBMCs which had been processed from human blood samples and preserved in freezing media (20% DMSO in RPMI 1640 media ) and stored in a liquid nitrogen cryofreezer. This work was done a couple of years ago by a former post-doc and not too effectively at that.

I've been attempting to thaw one patient's vial of cells and perform proliferation assays by first separating out monocytes for differentiation to dendritic cells. However, the number of starting cells immediately post-thaw is barely 0.5x10^6. I need to wash once, preferably twice, before I can even hope to select, and despite being as painstakingly careful as possible when aspirating off media by pipet, I still lose enough cells that I'm down to about 0.2x10^6; and after monocyte selection, I'm scarcely at 25,000 total--just enough for ONE replicate of dendritic cell differentiation and later proliferation with the next, and last, patient vial's 0.2x10^6 cells.

Is there any way I can retain more of my starting cells? We're spinning at 1200rpm for 10-15 minutes at 4C between washes. A better way to siphon off media? A better way to trap the pellet?

These samples are really precious and I dare not attempt another go with another patient's sample pair unless I'm sure I can retain more cells to make the assay worthwhile. Thanks in advance!

You might want to spin them down in 1.5ml microvials at 3000rpm for 3mins - 4C in a microfuge. Use P200 pipette to gently remove supernatant and leave behind 75-100ul of supernatant so as not to disturb pellet. Use gentle finger flicking to loosen pellet and add 1ml of wash buffer. Repeat the step. Try this and let us know how things go.