Cell Culture Techniques for Diverse Cell Lines

Mouse IMCD3s were grown in DMEM/Ham’s F12 50/50 (cat. no. MT10092 CVRF, Invitrogen) mix with l-glutamine supplemented with 10% fetal bovine serum (cat. no. S11150, Atlanta Biologicals) and 100 U/ml penicillin/100 μg/ml streptomycin (cat. no. 15140122, Invitrogen) in a 37°C incubator with 5% CO₂. PC12 cells were cultured as previously described (Newell-Litwa et al., 2007 (link)). Hek293T cells seeded in 35-mm plates were infected with 3 μl of lentivirus containing the shRNA constructs described earlier. After 24 h of being infected cells were incubated for up to 6 d in culture media supplemented with 4 μg/ml puromycin for selection as described in (Zlatic et al., 2011 (link)). Hek293 cells were then transfected with lipofectamine 2000 (Invitrogen) with 2 μg of DNA for 48 h.