Hi all,
Lately, I have been experiencing big problems while processing mouse spleens to obtain single cell suspensions. The aim of the work is to produce single cell suspensions, isolate CD4+ T cells using MACS sorting (Miltenyi) and set up some T cell assays. While a couple of months ago the first part of the procedure, that of obtaining large numbers of healthy splenocytes was working smoothly (5-10 x 10^7 cells/ spleen after RBC lysis and viability extremely high, I could hardly found any dead cells!!!), things have changed dramatically during the last few weeks. I am using the usual protocol, that is mash the spleen with the black rubber end of a 2 ml syringe through a cell strainer (I am using the 40 micron from BD) into a small petri dish together with plenty of medium (10% FCS RPMI 1640 supplemented with glutamine, pen+strep, mercaptoethanol and HEPES). At this very first stage and before lysing the RBCs, I use the trypan blue exclusion assay to have a look at the single cell suspension: Among a large number of healthy splenocytes, a considerable number (it varies from 8-20%) of large faint blueish cells are visible in the background: the vast majority of them are larger than the healthy cells and as i said before appear as light blue. i would ignore them but sometimes i find them in large numbers, especially taking into consideration their sudden appearance. I have tried everything: I have used unopened RPMI with no supplements just in case there is something wrong with them but no good. I have tried resuspending the cells in warm or cold media, using a petri dish or a 50 ml falcon nothing seems to work: these apparently dead large cells are always there.
I would really appreciate any comments on this issue especially if someone has experienced the same problem.
Thanks a lot
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