Tis epifluorescent microscope

H9c2 ventricular cardiomyocytes were purchased from ATCC (CRL-1446) and grown at 37°C, 5%CO₂, and 20% O₂ in DMEM containing 10% FBS and 1% pen/strep (GM) and changed every second day. When cells were ~75% confluent, 2×10⁴ cells/well were sub-cultured in 96-well plates for 24 hours before media was replaced with control media (CM; DMEM containing 2.5% FBS, 1% pen/strep) or LLC-conditioned media (LCM) as previously described[51 ],[52 (link)]. To generate LCM, LLC growth media was collected after two days of incubation in 162cm² flask with LLC density ending ~75% confluence. Media was centrifuged and filtered to remove cells and cell debris and diluted 1:4 (v/v) with serum-free DMEM. MitoTEMPO (MitoT; SML0737, Sigma-Aldrich, St. Louis, MO) diluted in PBS was added to CM or LCM at a concentration of 2 μM. After 2 hours incubation in respective media, 5μM MitoSOX Red (M36008, Invitrogen) in PBS was added to cells for 10 minutes, rinsed, and cells were visualized at 510/580nm (ex/em) on Nikon TiS epifluorescent microscope (Melville, NY) to assess mitochondrial superoxide production.

After 18 hours incubation in control or LCM with either vehicle or MitoT treatment, 5 μm MitoSOX Red (M36008, Invitrogen), a novel fluorogenic mitochondria-specific dye which on oxidation produces red fluorescence, in phosphate-buffered saline (PBS) was added to differentiated C2C12 cells for 10 minutes, rinsed, and cells were visualized at 510/580 nm (ex/em) on Nikon TiS epifluorescent microscope (Melville, NY) to assess mitochondrial superoxide production.