I am culturing SVG p12 cells in %10 FBS, %1 Pen/Strep DMEM medium. But whenever I passage them into a much larger flasks they are began to de-attached and started to die. I couldn't find any solution, does anybody know the reason?
Use scraper which is more convenient mechanical harvesting of the adherent cells than trypsin. At least one million cells/cm2 can be produced when growing cells as attached monolayers in culture (if the passage number exceeded 10). The average cell yields used with those sensitive cells are based on passage number and applying trypsin inhibitor. Actual cell yields can easily be several times higher or lower than this depending on viability percentage which can be calculated by trypan blue staining and culture conditions in your lab.
Hello Mothanna,
Firstable thank you for your reply. My SVG p12 cells was in T25 flask and their confluency was about 80%. I used 1 mL 1X tyripsin EDTA and incubated cells to de-attach for 5 mins at 37°C. After the incubation I added 2 mL culture medium as a trypsin inhibitor and collected cells. After couple of steps, I re-suspended and divided them into 2 T75 flasks. The passage number of the cells was 14. Why is that important?
Reduce the Trypsinization time. normally allow the trypsin to act and gently tilt the flask at a point of time you will see the cells separating one can use the fresh media and pipette the cells to suspension.
a 10% of spent medium can be added as the T75 flasks are bigger and after subculture the pH has to be better regulated.
this is late, but i just thought will leave this comment here.
Try with the following (Change the medium)
%10 FBS, %1 Pen/Strep, EMEM medium
Hopefully, it will work
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