I know that this varies between different labs and conditions, but I would like to hear your opinion in this matter. I am new to cell culturing, hence this question.
I our cell lab we work under sterile antibiotic free conditions and for culturing we use DMEM+FBS. From what I've heard PC3 should be confluent at about 8 000 000 cells in a T75-flask and the doubling time should be around 24 hours. What is your experience with this cell line? I feel that although splitting 2 milj. cells into a T75-flask, often they are not confluent after even 3 days.
Furthermore, we have big troubles counting the cells in a hemocytometer and the count very often turns out to be lower than what we expect from looking at the pellet or the confluency of the culture flask.
The normal duplication rate of PC-3 is about 36-40 hours which I have measured several times buy plotting the growth curve. Hence it takes nearly 6-7 days for a T75 flask to get fully confluent. About counting of cells, if you get a thick pellet the cells number should be quite high. Also the fully confluent flask gives millions of cells. Are you sure that the trypsinization brings all cells in suspended form. Some times trypsin gets weaker with time and takes longer time for cells to detatch from surface.
Thank you for your input! We have now done some experiments of our own and we found that the duplication time is about 30-31h in our conditions.
We also did a thorough study of our cell counting problems and counted the suspension, pellet and what was left in our T75 flasks during the duplication experiment. As it turns out, if too little solution is left in the T75 flask (although more than enough for the hemocytometer), then we get lower cell counts than expected. At least 0.5 mL should be left in the flask once the cells has been transferred to the centrifugation tube. We will now change the protocol so that we leave 0.5-1 mL in the flask when we split and thereby get better counting results, although it will mean discarding 5-10% of the cells. Furthermore, we now have a Countess that counts the cells for us :)
Morning, colleagues. Have started PC3 culture recently for anticancer drug testing. The MTT test with this cell line yields very liitle formazan (though, visible) and, hence, very low absorbance readings. Does anybody have an experience with this matter?
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