So here is the timeline:
Day 1: Cells received (shipped in culture flask from another lab)
Day 5: cells look healthy and happy (Pic 1)
Day 6: 100% confluent. Cells trypsinized with 1ml 0.25% trypsin, neutralized with 1ml medium and split 1:2 (i.e. one T25 to two T25 flasks)
Day 7: already 80% confluent. Cells trypsinized with 1ml 0.25% trypsin, neutralized with 1ml medium and transferred to T75. Added 13ml media (15ml in total).
Day 8: cells don't look great (pic 2)
Day 9: look worse (pic 3)
Is it because I didn't spin them down to remove trypsin before seeding the cells into a new flask? We have always been doing it this way and didn't have any problems... Any suggestions please??
Cells looking unhappy after splitting
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