Drosphila S2 cell culture - (May/16/2007 )
I am new in S2 cell cultures. I'm looking for a protocol to start a stable transfection of inducible GFP-constructs. I have recently began selecting a line with Hygromycin. I fail the first two times were all the cells died as soon as I split the culture. The third time all went well for a while. I could wean the cells from the selection antibiotic. I even subculture three times at high density in T25 (to keep them ). When I decided I had enough cells to try to expand to a T75 flask to freeze my stable cell line all went down hill. All the cultures died!. Imagine my frustration. Any suggestions on how to handle S2 cultures are much appreciatted as well as protocols for transfection and propagation!!