Grow cells in DMEM (Sigma #D9443) without L-Arg, L-Leu, L-Lys !
This DMEM is low glucose 1000mg/L so you will need to add and extra 3000mg/L to make it unto 4000mg/L in total. Thus 15ml of the 10% Glucose solution is needed for 500ml of DMEM.
Need to add 50ml of Mass Spec Grade Serum for final [conc.] of 10
1X Normal [Conc.] for DMEM media
L-Arg = 398 µM
L-Leu = 802 µM
L-Lys = 798 µM
From testing HeLa cells grow well and have very high incorporation rate at 1/4 to 1/8 of these levels. To play it safe we have been using 1/4. Thus:
L-Arg = 100µM (Light and Heavy)
L-Leu = 200µM (Only Light needed)
L-Lys = 200µM (Light and Heavy)
Grow cells for at least 5 doublings to ensure complete labelling. As a rough guide, if you Start on Monday, you will be ready by the following monday !