Penicillin streptomycin stock

Exosome-free fetal bovine serum (FBS) was generated from standard FBS (Cat. No. PS-FB1, Peak Serum) by ultracentrifugation at 120,000 g overnight at 4 °C. The supernatant was filtered through a 0.22 mm filter (Ultrafree-Centrifugal Filter Unit, Millipore), aliquoted into 50 ml tubes, and stored at −20 °C prior to use. SH-SY5Y neuroblastoma cells were grown in high glucose DMEM (Sigma-Aldrich) supplemented with 10% exosome-free (FBS) and 1% of a penicillin-streptomycin stock (catalogue number 30–001-CI, Fisher Scientific). Cells were cultured at 37 °C in humidified air containing 5% CO₂.
To inhibit autophagy, SH-SY5Y cells were treated with 50 nM bafilomycin A1 for 24 hours. To induce mitophagy, SH-SY5Y cells were treated with 1 mM DFP for 24 hours. For experiments utilizing both manipulations, cells were placed in 1 mM DFP for 24 hours, with 50 nM bafilomycin added for the final 16 hours. For the combination treatment, addition of the bafilomycin was delayed for 8 hours to ensure mitophagy initiation was able to occur [27 (link)].

Undifferentiated SH-SY5Y human neuroblastoma cells (ATCC, Manassas, VA) were cultured at 37 °C, 5 % CO2 in regular growth medium (pyruvate-free DMEM containing 25 mM glucose, purchased from Life Technologies, Grand Island, NY) and supplemented with 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) and 1 % of a penicillin-streptomycin stock (Fisher Scientific, Pittsburgh, PA). Medium was changed twice a day in order to minimize the effects of endogenously produced lactate. Cells were cultured to 60–80 % confluency in T75 culture flasks before use.