Mitoxpress xtra reagent

HEK293T cells were plated into two black 96 well clear bottom plates at 100,000 cells per well in 200 μl of DMEM 10% Fetal Bovine Serum. 8 hours after plating cells were treated as indicated for 16 hours. After the 16 hours, the media was changed 3 x with fresh media leaving a final volume 90 μl. The cells were equilibrated back to 37°C for 15min, after which 10 μl of prewarmed MitoXpress Xtra reagent (MX-200-4, Agilent) was added to each well. Prewarmed mineral oil was quickly layered on top of all analysis wells and the plate was measured over time using Perkin Elmer 2104 Envision plate reader at 37°C using time-resolved fluorescence measurement. 5-6 wells for each condition were analyzed (occasional wells with negative slopes were omitted) and the average rate (RFU/hour) was normalized to the cell count of three wells from the second 96 well plate.

HEK293T cells were infected with indicated pLVX-FEM1B-IRES-PURO viruses and selected for 1 day in puromycin as described in transfections and lentiviral packaging section. Cells were counted and plated into two 96 well black clear bottom plates at 100,000 cells per well (R126Q was seed 20% higher to compensate for proliferation defect) in 200μl of DMEM 10% Fetal Bovine Serum. The next day the media was changed 3x with a final volume 90μl, the cells were incubated for 10min at 37°C and 10μl of prewarmed MitoXpress Xtra reagent (MX-200-4, Agilent) was added to each well. Mineral oil was quickly applied to all analysis wells and samples were measured over time using Perkin Elmer 2104 Envision plate reader at 37°C using time-resolved fluorescence measurement. 6 wells for each condition were analyzed (occasional wells with negative slopes were omitted) per experiment and the average rate (RFU/hour) was normalized to cell count of three wells for each condition from the second 96 well plate (treated in a similar manner with 3 PBS washes).