Celltiter glo luminescence

Cardiomyocytes were seeded in 96-well tissue culture plates as previously described [21] (link). The cells were then exposed to eleven different conditions, namely 1 – media alone, 2 – conditioned media, 3 – HIV-1, 4 –100 µM of preferential caspase 8 inhibitor Z-IETD-FMK (casp8i) (Promega, USA), 5 –100 µM of preferential caspase 9 inhibitor Z-LEHD-FMK (casp9i) (Promega, USA), 6 – conditioned media and 100 µM Z-LEHD-FMK, 7 – conditioned media and 100 µM Z-IETD-FMK, 8 – conditioned media and 100 nM UCLA1 aptamer, 9 – HIV pre-incubated for 1 h with 100 nM of UCLA1 aptamer, 10 – HIV and 100 µM Z-LEHD-FMK, 11 – HIV-1 and 100 µM Z-IETD-FMK. The cells were harvested daily for 7 days followed by quantification of the caspases and cellular ATP levels, using the Caspase-Glo8, Caspase-Glo9 and the CellTiter-Glo luminescence-based detection kits as instructed by the supplier (Promega, USA). Wells containing media alone were used as controls for background luminescence and subtracted from the test values. The results were expressed as relative light units (RLUs) and plotted in a graph relating RLU to number of days.

mitochondrial ATP was determined by CellTiter-Glo luminescence (Promega). Standard ATP was used for measurement of ATP content (nmol/mg protein) in control mitochondria or AM-mitochondria.

Adenosine triphosphate (ATP) level was determined by CellTiter-Glo luminescence (G7570, Promega, USA). For intracellular ATP content, cells were incubated by 200 μl reagent buffer each well, standing for 30 min at room temperature for lysing cells. For extracellular ATP, the culture medium was collected and centrifuged at 2000×g for 10 min. Then, the supernatant was collected and centrifuged at 20,000×g for 20 min at 4 °C, with the remaining lower half to use. CellTiter-Glo luminescence test solution (50 μl) was added into culture media (50 μl) and incubated for 30 min at room temperature in opaque-walled 96-well plates. Luminescent signal was determined by the microplate reader.

Extracellular adenosine triphosphate (ATP) was determined by CellTiter-Glo luminescence (#G7570, Promega, Madison, WI, USA), which can perform cell lysis and generate a luminescent signal that is proportional to the amount of ATP present. Basically, opaque-walled 96-well plates with CM (50 μl) or mdCM (50 μl) were prepared. Then, 50 μl CellTiter-Glo luminescence test solution was added and incubated for 30 min at room temperature. Luminescent signal was recorded using a luminescence microplate reader (BioTek Instruments, Winooski, VT, USA). Since mitochondria produce and restore most of the cellular ATP, the number of released mitochondria in the CM can be estimated by evaluating the extracellular ATP.