In my current project I am planning to isolate mononuclear cells from peritoneal ascites of cancer patients using ficoll centrifugation. However, following isolation of the mononuclear cell layer, I would still be keen to freeze the remaining cells in the pellet (tumour cells, stromal cells, granulocytes etc). Is there any problem with resuspending the pellet, washing the Ficoll off, lysing RBCs and then freezing?
Thanks,
Gabriel
@Gabriel Osborn
There should be no problem with that. Resuspend the pellet and wash 1-2x with PBS abundantly in order to remove the Ficoll. Then resuspend the cells in freeze media and you're ready to go.
As for RBC lysis, as with any additional processing, this is stressful for the cells. Is your ascites fluid haemorrhagic to such an extent that you need to lyse the RBC? If you only have traces of RBC, maybe it'd be better to skip this step.
Anyway, i suggest you run some small experiments and decide for yourself how the protocol is going. Practice beats theory any time of the day. Good luck!
Good day! I isolated PBMCs from whole blood on a ficlol gradient, assessed cell survival, and frozen them. It is necessary to apply ficcol very carefully so that there is not a single drop on the tube. It is necessary to very accurately maintain the time and conditions of centrifugation (with ficoll - 10 min at 20C, 1000g). When adding a cryoprotectant, it is necessary to centrifuge in the cold at 4 C, 300g) The survival rate and the number of cells greatly depend on these conditions.
Hi Gabriel,
It is no problem, I perform two washes with saline solution (0.9% sodium chloride) mixing softly!! Then centrifugation 300g for 10 min, and after the last wash, carefully remove supernatant completely and resuspend cell pellet in RPMI medium with 10% DMSO, as cryoprotectant.
I see that Alexandr and Maria have botj given a recipe for freeze medium above and i would like to add that post-thaw viability is increased if the freeze medium also has FBS.
Our recipe for freeze media is
- RMPI (supplemented with 1% antibiotic/antimycotic solution)
- 20% FBS (10% is fine though)
- 10% DMSO.
Anyway, i have seen each laboratory has it's own recipe so as long as it makes you happy, it's fine.
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