My plan is to isolate immature myeloid cells (IMCs) from the spleen and to expand them into MDSCs with IL-6 and GM-CSF supplemented media.
I have successfully isolated splenocytes from tumor-bearing mouse spleens during late-stage tumorigenesis. Over 500 million live cells were isolated from a 1-gram spleen. The cells were frozen in cryogenic tubes - 50 million cells in 1 mL freezing media (with 10% DMSO/70% FBS).
Two months later, 25e6 cells were thawed in supplemented RPMI 1640 media containing 10%FBS, 55 uM BME, 1 mM sodium pyruvate, 25 mM HEPES and 200 mM L-Glutamine.
I noticed that 24 hours after thawing these cells the viability was very low - I collected both (1) the cultured media and (2) the trypsinized and neutralized cells in a conical tube, centrifuged them at 1200 rpm, then re-suspended them and counted with trypan blue to find 1e6 live/0.750e6 dead cells.
Do you recommend freezing the splenocytes after isolation, or should the cells be placed into culture immediately after isolation?
Thank you, and sincerest regards.
The vial was simply warmed in a 37 C water bath for 1 min. I found out that freshly isolated MDSCs are most viable and reliable. Tumor-bearing mice generate almost 90% PMN-MDSCs (which are very similar to neutrophils), and they will not survive freeze thawing: Article Myeloid-derived suppressor cell measurements in fresh and cr...
For my experiments, I use patient primary isolated cells (I isolate the two subsets). In my experience, 85% of G-MDSCs die almost immediately after thawing, but M-MDSCs stay alive for longer. You should also check their functionality, because for example even though M-MDSCs stay alive longer, they no longer have the ability to suppress T cell function.
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