Dear All,
i would like to ask why my negative controll in cell culture fails from time to time. I am performing simple cytotoxicity assays based on Balb3t3 and L929. I am seeding the cells on 96-well plate and after 24 hrs i change the media into test samples. I change also the negative control for the fresh medium (with 10% of serum) and vehicle control (with 5% of serum, since my testing compounds are dissolved in lower-serum medium). And sometimes (very rarely, but there are the incidents) it happens that the cells in my negative control look disgusting. Can anyone help me with it?
Katarzyna Pocheć You have to maintain the same concentration of serum as you maintained in test samples. The serum contains growth factors, so maybe your negative control showing growth as compared to test samples.
Try this it will work.
Dear Sameer Varma thank you for the answer but i am not sure if the serum concentartion is the main reason here. If the vehicle control (with low serum) looks perfectly fine under the microscope and the negative control (that is cultivated with full serum - 10%) is damaged then i am afraid it must be a different reason.
Dear Katarzyna Pocheć I don't know what is your initial cell count in each well, if in negative control you are using 10% serum, so there may be overcrowding which leads to cell death. I always maintain 2% concentration in every well (test as well as control), so that concentration is not enough to grow but enough to survive.
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