Apoptotic versus non-apoptotic cells were then counted using Imaris software.
Extended Data Fig. 1 cGAS translocates to the nucleus in non-apoptotic cells with intact nuclear membranes.
Quantification of radiation-induced protein expression levels was determined in non-apoptotic cells only by the image analysis software program48, such that advanced apoptotic cells observed as a gross change in area and morphology of the DAPI-labeled nuclei, were not included for analysis.
The differential uptake of these two dyes allows the identification of apoptotic and non-apoptotic cells (Babykutty et al. 2009).
Live/dead assay using ethidium bromide/acridine orange cocktail on fibroblast cells grown on the hydrogels after 5 days of initial seeding displayed green nucleus revealing the non-apoptotic cells.
This contrasts with the untreated non-apoptotic cells, which showed a low fluorescence, smooth, flattened nuclear morphology and normal nuclei, as well as uniformly dispersed chromatin [Fig. 2c (i, ii)].
In non-apoptotic cells with intact mitochondria, JC-1 selectively enters the mitochondria where it aggregates and exhibits a fluorescence emission at 590 nm.
Notably, non-apoptotic cells with externalized PS can also be taken up by macrophages and suppress ROS and RNS production [19].
Direct fluorescence comparison between apoptototic and non-apoptotic cells is difficult due to their different cytosolic volume, thus we followed on this observation by investigating whether silencing BAG3 had an effect on apoptosis.
To correlate the apoptosis with the proinflammatory phenotype, the proportion of IL-8 secretion to the percentage of non-apoptotic cells was measured in AM transfected with CFTR-siRNA or control-siRNA.
As shown in Fig. 3A, Lexa and CHX combination treatment induced ROS dramatic increase in apoptotic cells, but not in non-apoptotic cells, suggesting ROS may contribute to CHX-and Lexa-induced apoptosis.
