Hello,
I recently started culturing a suspension cell line (L-428, Hogdekin lymphoma). I thawed a cryo-vial of the cell line, added pre-warmed RMPI + 10%FCS + Pen/Strep, centrifuged at 800 g, removed the media and added fresh, pre-warmed one and finally seeded them at 0.5 x 10^6/ml in 6-well tissue culture plates. During the first days they seemed quite ok, on day 3 they appeared as clumps (see picture L-428_Day3) and on day 4 I exchanged media. Since Day 7 they look more or less the same: not really smooth, but somehow eneven with a high "granularity" (see picture L-428_Day15). We changed the media to RPMI + 20% FCS + PenStrep hoping that this might do the trick and make them divide, but nothing changed. So the cell count doesn´t change. Acc. to trypanblue staining half of the cells are dead, the other half vital. Mycoplasma test (PCR) is negativ.
Has anybody run into similar troubles? Or does the picture gives anyone a clue what we might have done wrong? I would appreciate every suggestion!!
Regards,
Agnes
Also make sure the P/S concentration is 1%. Some cell death is okay. Can you post a picture of your cells in day 2 in culture here. Cells may not need 20% FCS/FBS.
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