How to prevent HL-60 cell contamination? 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. Thanks

The single most likely reason for the contamination is that your frozen stock is contaminated. Assuming you've been properly trained in cell culture, you're working in a correctly-maintained biosafety cabinet, your reagents are all sterile (has your media been sterile-filtered after adding FBS, etc.?), and the incubator is clean (water bath pan has been autoclaved, sterile water with biocide/fungicide added to it, entire incubator wiped down and all removable parts autoclaved periodically, etc.), then stock contamination is the most reasonable answer. Try thawing earlier passages than you have been working with, or order a new vial or two from ATCC or whoever your cell vendor is. Make sure your pipetors are clean, and you're using sterile filtered pipet tips.