13c615n4 l arginine hcl

AC16 cells procured from Millipore between passage numbers 11 and 16 were cultured in DMEM/F12 supplemented with 10% FBS and no antibiotics. Cells were maintained at 37 °C with 5% CO₂ and 10% O₂. For isotopic labeling, SILAC DMEM/F12 (Thermo Scientific) deficient in both L-lysine and L-arginine was supplemented with 1% dialyzed FBS and heavy amino acids ¹³C₆¹⁵N₂ L-Lysine-2HCl and ¹³C₆¹⁵N₄ L-Arginine-HCl (Thermo Scientific) at concentrations of 0.499 mM and 0.699 mM, respectively. Light media was switched to heavy media, and cells were labeled for 16 h prior to harvest. UPR was induced with 1 µM thapsigargin (SelleckChem) or 1 µg/mL tunicamycin (Sigma) at the same time as isotopic labeling.

Eight B-cell lymphoma cell lines (DSMZ, Braunschweig, Germany) were selected for generating the super-SILAC reference sample; SC-1, DoHH2 (transformed follicular lymphoma), OCI-LY3, U2932 (non-GCB DLBCL), SU-DHL-5, SU-DHL-6, SU-DHL-10, and SU-DHL-4 (GCB DLBCL). The cell lines were cultured in RPMI (Thermo Fisher) and penicillin/streptomycin (P/S) with either 10% fetal bovine serum (FBS) (OCILY3, SC-1, DoHH2 and SU-DHL-4) or 20% FBS (SU-DHL-5, SU-DHL-6, SU-DHL-10) supplemented with heavy ¹³C₆ L-Lysine-2HCl (Thermo Scientific, prod #88431) and ¹³C₆¹⁵N₄ L-Arginine-HCl (Thermo Scientific prod #88434). All cell lines were tested negative for mycoplasma. Cells were cultured for approximately 10 cell passages to allow maximum incorporation of the labelled amino acids in all proteins. [15 (link)–16 ] The incorporation was checked with mass spectrometry for each individual cell line. After confirmation of sufficient incorporation, cell lines were lysed as described above and 20-fold concentrated with the Vivaspin^® 2 Centrifugal Concentrator. The final protein concentration was measured using a BCA protein assay.