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Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar

ody was added to the plate, with the final concentration per well ranging from 400 ng/mL down to 0 ng/mL of antibody. The plates were then incubated at 37C / 5% CO2 for a total of 5 hours to induce apoptosis. 3 hours into the 5 hour treatment, CellTiter-Blue reagent was added to each well with the Equator (note: for plates later receiving Caspase-Glo 3/7 reagent, CellTiter-Blue was diluted 1:4 in 1X PBS before addition to the assay plate). After the CellTiter-Blue addition, plates were incubated for 2 hours at 37C / 5% CO2. When the 5 hour incubation with anti-FAS antibody was complete, fluorescence was recorded at excitation 540 nm and emission of 590 nm with the PHERAstar. The caspase reagents were then added to the plates with the Equator. Apo-ONE was added to one plate containing CellTiter-Blue reagent, and the plate was incubated at room temperature for 1 hour, followed by fluorescence reading with the PHERAstar at excitation 485 nm and emission of 520 nm. Caspase-Glo reagent was added to the plate receiving the diluted CellTiter-Blue reagent, and incubated for 1 hour at room temperature. Luminescence was then recorded with the PHERAstar.

For the 1536-well assay format, a density of 4,000 cells per well was plated with the Deerac Fluidics Equator. The remaining multiplex protocols were performed identically to the low-volume 384 protocols listed above.


Results and Discussion
Promegas CellTiter-Blue assay multiplexed with either Caspase-Glo 3/7 (figure 2) or Apo-ONE assay was prepared in both low-volume 384- and 1536-well format (f
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