Hello

I would really appreciate any advice on this, no-one in my institute has any suggestions for what I suspect is a common problem for beginners.

I would like to prepare PBMC from normal fresh human bone marrow. I plan to freeze it down and then facs all the samples together when I have enough. My problem is that when I look by flow cytometry at a small aliquot after lymphoprep, 50% of the cells in the sample are in the high side scatter region, which I believe are granulocytes. I understand that on thawing the samples after freezing down with 10%DMSOin FCS, granulocytes do not survive well and die off, releasing their granular contents and negatively affecting the bone marrow progenitors I am interested in.

My question is, what can I do to remove these granulocytes? I think I must be doing something wrong in my lympoprep. I've attached a summarise version of my protocol below:

1. Dilute BM aspirate with sterile ice-cold RPMI-10% FCS to a total volume of 12ml.

2. Perform Lymphoprep separation with the aspirate sample. – Prepare two 15ml tubes, and add 3 ml of ice-cold Lymphoprep into each 15 ml tube and carefully layer 6 ml of the dilute bone marrow aspirate over the Lymphoprep. Centrifuge at 600 x g, 30 min, 4 ^oC without acceleration and with brake off.

3. Collect the interphases containing mononuclear cells, pool the interphase Mononuclear cells, and wash pooled interphase Mononuclear cells with RPMI+10%FCS (300 x g, 4C, for 10 min). After washing resuspend MNC pellet in FCS by gently pipetting up and down.

4. Count living cells (20 ul) using Trypan blue staining solution using a Neubauer hemocytometer or cell counter and calculate the total MNC number for the sample.

5. Proceed to freezing.

I would be very grateful for any advice or tips in resolving this problem.

Thanks for reading this!