Adenoviral Vector Transduction of R28 Cells

R28 cells were purchased from Kerafast, Inc. Boston, MA. R28 cells from passages 67 to 70 were cultured in DMEM with low glucose (Sigma, cat# D5523) supplemented with 10% fetal calf serum (Cytiva HyClone, cat# CSH3007303) and 1% penicillin-streptomycin (Gibco, cat# 15070063), 7.5% sodium bicarbonate (Sigma, cat# S8761), 1% MEM non-essential amino acids (Gibco, cat# 11130051) as described previously [33 (link)]. R28 cells were differentiated overnight in complete medium supplemented with 250 μM pCPT-cAMP (Sigma, cat# C3912) on laminin-coated plates. The next day, cells were transduced with adenoviral vectors containing either A2 or RFP for 6 h at 5, 10, and 20 multiplicities of infection (MOI), as previously described [7 (link)]. The final MOI of 20 was chosen according to previously published studies and preliminary experiments [7 (link)]. After 6 h, the media was changed to normal complete growth media for 24 h. The following day, cells were treated with vehicle or 1 µM ABH or 5 mM L-Glutamate for 16–18 h and subjected to Western blot and Seahorse XFe flux analysis.