Which cell line for Cell cycle analysis - (Jul/12/2007 )
I want to overexpress my protein of interest in a human cell line and analyse the cell cycle profile post transfection. My choices of cell lines are HEK293, HEK293T, COS7 or Hela cells. Any suggestions as to which cells I should use and what time points to analyse. I have briefly tried the experiments in HEk293 cells but I dont see any difference in cell cycle profile by FACS at 24 or 48 hours post transfection. However, when I examine overexpressing cells by immunofluorescene there doesnt really appear to be any cells in mitosis BUT there appears to be no difference by FACS analysis. Should I be analysing the FACS data earlier or maybe I could be loosing my mitotic cells after fixation for microscopic analysis (I use methanol to fix)? There does however appear to be a small number of cells in the G2/M range after FACS analysis (only about 5-10%) and I am wondering if this cell line is a bad choice. I am new to this so I am just looking for some general suggestions. Thanks very much!
Have you checked that your protein is really
being overexpressed post-transfection by Western or immunoflurescence?
This is important. Also, transfecting and checking cell cycle isn't really
a whole lot of work so I would suggest trying it in as many
cell lines as possible as the pathway your protein is working in
may be disabled in some cell lines.