Hello Alfin Mohammad Abdillah ,

Your cells are either contaminated or under stress conditions.

There may be mycoplasma or yeast contamination.

There are various methods or kits are available to detect contamination.

Some points could be helpful like...

• If the cells are looking fine after reviving then replace the PBS and Trypsin for subculture. Maybe your PBS or Trypsin is causing the trouble.
• Change the stock of media and FBS which is being used during the cell culture.
• If you are facing the same issue then you need to use the cell stock of different time points. Maybe the cells were contaminated during preservation time and after revival, the contaminant is growing. So revive a frozen vial and repeat the same.

All the best

Dear Nilesh Khandelwal ,

Thank you for the response, for now I already replace all PBS and Trypsin, and for media I still use the new one.

And interestingly I use some aliquote from the contaminated plate before for subculture in media with 3X anti-anti and the cell comeback normal.

And for another experiment I also starting from the frozen stock growing it in usual media and for subculture become 3 plates, I used 1X, 2X, and 3X anti-anti concentration. And the results, cell growing normal but in 1X anti-anti there is some small black spot that I'm not sure it is contamination or just dead cells.

Are the media with high concentration of antibiotic will influence the cells, differentiation, etc?

Thank you

Dear Alfin Mohammad Abdillah

It is good to know that your cells are growing fine in a culture containing 2X and 3X antibiotic solutions.

I have not tried yet the same but some of the research articles are showing that the higher concentration also affects the differentiation process and experiments.

Here are some examples:-

Antibiotics in Cell Culture: Friend or Enemy? - PromoCell

Side Effects of Culture Media Antibiotics on Cell Differentiation (PMID-26037505)

So If needed you can increase the concentration in PBS but not in the culture medium. If the reason is contaminated cells then you have to take the fresh batch of cells or exclude the reason for contamination.

One more thing, if these are dead cells only so they will be removed after one PBS wash so try the PBS wash.

Thank You and All the best 👍

Dear Nilesh Khandelwal ,

Yes thank you for the suggestions, for now I'll monitor the cells under 2X antibiotics condition. And the next I'll try to use antibiotics in PBS and Trypsin.

Thank you :)