Dmem with glucose and l glutamine

NIH-3T3 mouse fibroblast cells (ATCC, CRL-1658) were cultured in T25 NuncTM flasks (ThermoFisher Scientific) as in ^{31 (link)}. Cells were incubated and suspended in growth media (DMEM with Glucose and L-Glutamine, Lonza, 12–604F) with 10% Calf Bovine Serum (30–2030, ATCC) and antibiotics (Penicillin Streptomycin, 100ug/ml) at 37 °C and 5% CO₂. For sample preparation, cells were seeded in growth media (DMEM with Glucose without phenol red, Lonza, 12–917F) with 10% Calf Bovine Serum (30–2030, ATCC) and antibiotics (Penicillin Streptomycin, 100ug/ml) at concentration 35,000 cells/ml overnight in petri dishes (MatTek, P35G-1.5–20-C). After 20–24 hours, cells were transfected using Lipofectamine 3000 (L3000008, Invitrogen) with 2 μg of total DNA (1μg of HA-Dendra2 and 1μg of PAmKate-PH) and treated with Control (0 μm CPC + Tyrodes- BSA vehicle), 5μM and 10 μM CPC for 1 hour in the incubator under same conditions. After one hour cells were washed twice with phosphate-buffered saline (PBS) (Sigma-Aldrich, D8537), fixed at room temperature in 4% paraformaldehyde (PFA) (Alfa Aesar, J61899AK) for 10–15 minutes, and then washed again with PBS two times.