I have thawed Hl-60 cells from liquid nitrogen chamber two times in RPMI 1640 media (containing 10% FBS, L glutamin, penicillin/streptomycin). In the first day the cells remain okay. But when subcultured from the first flask to new flasks (cell solution : media= 1:3) all the flasks became contaminated on the 2nd day of culture. Cloud was found in both time. It can be mentioned that I used sterile one-time usable pipettes and uncoated flask for growth. All the transfers to other flasks and media addition were done very carefully and done inside clean bench. Before using clean bench, it was sprayed by 70% EtOH.
Has anyone experienced like this problem? Please share and if have, please give some valuable suggestions or references.