Hi everyone,
I have been culturing HEK293T cells that have been engineered to overexpress the mechanosensitive protein Piezo1. However, I am finding it very difficult to keep them in culture - even though they are pretty standard cells - they constantly get cell contamination, even with 1% Pen/strep mix in their media.
The other day, I found that they had cloudy media and that there seemed to be moving bacteria inside with the cells? (as seen in the uploaded video). Please let me know if you have any idea of what this might be and if you have any suggestions of what I might do to avoid this in future cell culture experiments.
Media used: high glucose DMEM + L-Glutamine + Sodium Pyruvate + 10% FBS + 1% Penicillin/Streptomycin
Contamination when there are antibiotics in your media might suggest you have a big contamination source. That might be your incubator, or the vacuum hose, or even your cabin. I hope you have clean stocks for these cells. I would suggest discarding these cells, sterilizing everything and start with a new batch.
Hello Mariana
If you feel that the contamination is bacteria-like you should confirm it on LB agar plate. Make a streak on the agar plate with the contaminated medium and place it at 37 deg overnight. Observe the plate for bacterial colonies the next day. Please do not keep the contaminated culture in the incubator for long as there is a possibility that other cultures could get contaminated.
If you are facing the contamination problem often I suggest you fumigate the culture room over the weekend. Also, clean the CO2 incubator with 70% alcohol and place autoclave sterile water in the tray placed at the bottom in the incubator. You can also try autoclaving the trays of the incubator.
I would suggest you thaw a fresh vial of cells after having done all the cleaning.
If you feel that you can get rid of the contamination you can try using Normocure instead of Pen/strep. But I prefer you start fresh because contaminated cells have their metabolism changed.
Good Luck.
There is a probability you are using a contaminated stock of cells. Try using stocks from a different source.
If you are using a communal incubator, I highly recommend taking a look to see if anyone has left any cultures in for a long time and forgot them. I had an experience a few years ago where I found two T-75 flaks in the back of one of our communal Co2 incubators that had been forgotten about by someone for over 3 months and had both grown into minature biomes...
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