For SILAC, HeLa cells were grown in “light” and “heavy” SILAC DMEM (Thermo Fisher Scientific, #88364), supplemented with either l-arginine, l-lysine, or their heavy isotope counterparts l-arginine (13C6, 99%; 15N4, 99%; Sigma-Aldrich, 608033) and l-lysine (13C6, 99%; 15N2, 99%; Sigma-Aldrich, 608041). Arginine and lysine were added at the concentration of 84 and 146 mg/liter, respectively. The SILAC media were further supplemented with 10% dialyzed FBS (Gibco, Life Technologies, 26400-044), 1% glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), and 10 mM Hepes (pH 7.5).

For hmSILAC, HeLa and NB4 cells were cultured in light and heavy hmSILAC DMEM media (Carlo Erba reagents, custom-made), whereas the ovarian cancer cell line SK-OV-3 was grown in hmSILAC RPMI media. Both custom-made media were supplemented with l-arginine (Sigma-Aldrich, A6969), l-lysine (Sigma-Aldrich, L8662), and either l-[13CD3]methionine (Met-4, heavy, Sigma-Aldrich, 299154) or l-[12CH3]methionine (Met-0, light, Sigma-Aldrich, M5308), respectively. The concentration of l-methionine was 30 mg/liter. The hmSILAC media were then supplemented with 10% dialyzed FBS (Gibco, Life Technologies, 26400-044), 1% glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), and 10 mM Hepes (pH 7.5).

Both SILAC and hmSILAC HeLa cells were grown in the respective heavy isotopes containing media for at least nine replication cycles to ensure full incorporation of heavy amino acids, with careful monitoring of their growth rate, viability, and overall morphology, to guarantee that the normal physiology was preserved.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.