Hi everyone,
I’m working with the MDA-T120 thyroid cancer cell line (BRAF V600E). I thawed a vial yesterday and seeded into a T25 flask (5 mL medium), but after ~24 hours most of the cells are still round and floating, with only a few starting to look spindle-shaped. The medium is still red (pH stable).
Here’s what I did:
- Thawed the vial quickly
- Centrifuged at ~400×g for 5 minutes
- Resuspended in ~5 mL complete medium (RPMI-1640 + 10% FBS + L-glutamine + NEAA)
- Seeded into a T25 flask and incubated at 37 °C, 5% CO₂
Does anyone have tips for improving post-thaw attachment or recovery for this line? Would it help to increase FBS concentration or adjust seeding density?
Thank you!
It’s hard to say about seeding density unless you state how many cells were in the vial.
Did you check viability of the floating cells by taking out an aliquot and checking with Trypan Blue? If floaters are dead, you just may have a bad freeze or they don’t like centrifugation right after thawing. You can slowly dilute freshly thawed cells with 9 ml completed media and seed your flask, changing media the next day. If they are alive, you can try treating your flask with poly L lysine reagent before plating.
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