help: macrophages transfection and immunofluorescence - (Oct/28/2005 )
hello, in fact I've two problem to resolve:
-first, I want to transfect primary macrophages, wich transfection method can I use?
-and, I ever did a first transfection test (using the GFP) with fugene, lipofectamine and phosphate calcium, but there's a lot of autofluorescence. I fixed the cells with PFA 4%, I blocked the PFA by treatment with PBS/glycine, and I treated with ammonium chloride in order to decrease the autofluorescence, and I did all the PBS wash between all the steps. and finally, the coverslips was mounted in mowiol. (fresh mowiol)
If somebody have good tricks for these transfection and autofluorescence problem It would be very helpfull for me.
primary macs and dcs are really hard to transfect (using conventional means generally never higher than 10%). In our lab we use two different systems-lentivirus and amaxa. Both give us good efficency with minimal death.
Also, in general macs and dcs just have high autoflourescence. we use a cy5 label for figuring out transfection efficency and it works nicely (i like it better than GFP).
Hope this helps
try with PEI or products from Polyplus Transfection derived from this molecule...