2.4. Cellular respiration assay

XF24 extracellular flux analyzer from Seahorse Biosciences was used to measure the rates of fibroblasts oxygen consumption, as previously described [11]. Cells were plated the previous day of experiment on the XF24 cell culture microplates at a density of 60,000 cells per well. XF24 cartridge was equilibrated with the calibration solution overnight at 37°C. XF assay media (5 mM glucose, 2 mM Pyruvate, in DMEM (Seahorse Biosciences) was prepared and pH adjusted to 7.0 on the day of the experiment. XF assay media was used to prepare cellular stress reagents, 500 nM Oligomycin, 500 nM FCCP, 100 nM Antimycin and 100 nM Rotenone (final concentration). All the reagents were loaded in the ports as suggested by Seahorse biosciences. Oxygen consumption rates were measured for 3 min with 3 min of mixing and 2 min of waiting period. After the assay was completed, cells in each well were counted using ViCell cell counter and the counts were used to normalize the OCR rates. OCR was expressed as nmoles of oxygen/min/1000 cells.