Carbonyl cyanide 3 chlorophenylhydrazone cccp

HSPCs were harvested from expansion cultures at day 7, resuspended at 1 × 10⁶ cells/mL in RoboSepTM Buffer and incubated with 2 µM MitoProbeTM JC-1 cationic dye (Thermo Fisher Scientific; dissolved in DMSO) at 37 °C/5% CO₂ for 30 min as per manufacturer’s guidelines. For control sample, 50 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Thermo Fisher Scientific) was added simultaneously. Cells were washed, resuspended in RoboSepTM Buffer, and stored on ice until analysis. Samples were analysed on a Guava^® easyCyte Single Sample flow cytometer, using 488 nm excitation with 530/590 nm emission filters. JC-1 is a lipophilic, cationic dye that can selectively enter mitochondria and reversibly change colour from green to red as the membrane potential increases. In healthy cells with high mitochondrial (ΔψM), JC-1 instinctively generates complexes known as J-aggregates (oligomers) with red fluorescence. Whereas, in unhealthy or apoptotic cells with low (ΔψM), JC-1 maintains its monomeric form with a green fluorescence.

Flow cytometry can be used to evaluate changes that occur in cells over short periods of time. Biological activities such as change in calcium content in response to drugs; the generation of reactive oxygen species or mitochondrial membrane changes during apoptosis and phagocytosis rates using labelled bacteria, can all be assessed. To determine mitochondrial membrane potential in the cells, the MitoProbe DiIC1(5) kit (Thermo Fisher Scientific, Waltham, MA, USA) was utilised. In eukaryotic mitochondria, DiIC1(5) accumulates in relation to mitochondrial membrane potential. The cells were plated at a density of 1 × 10⁶ cells in each well in 12-well plates and treated with complete media for 24 h. The cells were treated with 10 µM of 1,1′,3,3,3′,3′-hexamethylindodicarbo-cyanine iodide (DiIC1(5)) after incubation. Cells for negative control were cultured at 37 °C with 5% CO₂ for 15–30 min after being treated with 50 µM Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Thermo Fisher Scientific, Waltham, MA, USA), a protonophore mitochondrial toxin. The cells were given a wash with PBS after incubation before being examined with a flow cytometer. The median fluorescence intensity was used to calculate the data (MFI).