I need to freeze Mononuclear Bone Marrow cells and thaw them at a later timepoint to analyse them via FACS. To freeze the cells I use 90%FBS and 10%DMSO. But when I thaw the cells (using RPMI,10%FBS) and wash them (with PBS), the pellet sticks together and I can't dissolve it to distribute the cells evenly into different wells.
Does anyone have an idea how to prevent the cells from clumping togehter?
I really appreciate any input, thank you in advance.
Hello Mats Urban
The freezing medium which you should use is (90%FBS +10% DMSO) or ( 90% culture medium containing FBS + 10% DMSO). The freezing medium which you are using (DMSO +10%FBS) is not right. How much of DMSO are you adding to the freezing medium? Is it 90% !
Best.
Hello Malcolm Nobre ,
excuse my mistake, I am using 90%FBS, 10%DMSO as freezing medium.
I changed the question accordingly.
Mats Urban , what is the viability percent on thawing? Have you taken a cell count after thawing?
I would suggest that the cells stick together because a lot of debris is produced in the course of the freeze-thaw cycle. The DNA often acts like a glue when it leaks out of cells.
I wonder If you could add DNAse in the wash buffer. Or may be treat them with Accutase before seeding them into the wells.
Another option is to wash the cells not with PBS but with FBS and at low speed - something like 70g for 10 min - that would sediment healthy cells prior to debris.
By the way, are you going to culture these cells prior to FACS or you intend to stain them right after the thawing? If the latter, then you could fix them with 2% PFA right after the the first wash. That would diminish the clumps formation greatly.
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