it are considered to be the end-product of intracellular nitroimidazo

Quantification and automatic imaging of caspase activation using the DNV fluorogenic substrate Cells were dispensed in 45 uL medium in 384 well microplates using the Multidrop 384 dispenser Afatinib and incubated within the Steri Cult incubator. If pre treatment with Z VAD FMK pan caspase inhibitor was tested, it was performed 1h just before treatment in a final concentration of 40 uM in hands down the DMSO. Transfection with siRNAs or treatment with small molecule was done 24h post cell seeding by moving siRNA buildings or medicine dilutions from a polypropylene 384 well source plate to the 384 well assay plates using the PP 384 M Personal Pipettor, after which 5 uL of 5 uM DNV answer in PBS were dispensed to the assay plates using the FlexDrop IV. As explained above, over a time length of up to 96h post DNV substrate addition Images were acquired around the INCA0. Each analysis condition was performed in duplicate and reported data refers to the average of two wells, aside from RNA knockdowns studies which were performed Lymph node in quadruplicate; reported data in that case match the average of four wells and error bars represent the standard deviation of the data obtained in quadruplicate. For comparison of the NucView488 signal with nuclei count, DRAQ5 live staining of nuclei was performed after the last timepoint by adding DRAQ5 towards the cells diluted in PBS to achieve a final concentration of 2. 5 uM. As described above pictures were acquired to the INCA0. siRNA transfection Cells were seeded in 384 well microplates as descrived above and transfection with GFP or cell death siRNA pool was performed 24h article cell seeding. Transfection of cell death siRNA pool in HeLa Empty checkpoint inhibitors cells described in Figure 3 was done using 0. 025 uL Lipofectamine RNAi Max per well; siRNA transfection in HeLa Empty and HeLa Bcl XL cells described in Figure 6B was done using 0. 075 uL Lipofectamine 2,000 per well. siRNAs were preincubated with the transfection reagent for 20 minutes at room temperature in OptiMEM, and 10 uL of the complex were utilized in the assay plates. The siRNA final concentration was nM for all transfections. Following transfection, 5 uL DNV substrate solution in PBS was added to each well utilizing the FlexDrop IV, and as described above 48h post transfection automatic imaging and quantification of caspase activation was performed. Review of the effect of the DNV substrate around the proliferation of HeLa Bcl and HeLa cells HeLa Empty XL cell suspensions were seeded as explained above; at 24h post seeding, 12-point doubling dilutions of the DNV substrate in ten percent DMSO starting from 0. 5 uM to at least one mM were prepared in a polypropylene 384 well microplate, and 5 uL of each dilution were transferred to the assay plates to achieve one last concentration of DNV substrate ranging from 0. 05 to uM in 10 percent DMSO. The assay plates were incubated for 24, 48, 72 and 96h inside the Steri Cult incubator.