DAPI-based Cell Viability Quantification

Cell survival was assessed by DAPI nuclear staining. This method to distinguish physiologically intact from dead or dying neurons was validated by comparison with testing membrane integrity with the trypan blue exclusion assay [49 (link)] and TUNEL staining [this study]. Fixed cells were washed three times in PBS before washing twice with PBS/0.1% Triton-X-100 (PBST). Cultures were incubated for 30 min in DAPI (Sigma-Aldrich; Munich, Germany; 1:1000 in PBST) in the dark. Subsequently, coverslips were washed five times in PBS and once in DABCO (Roth, Kalsruhe, Germany) before mounting in DABCO. Experimental groups were evaluated with an epifluorescence microscope (Zeiss Axioskop; 40 × objective, Oberkochen, Germany) equipped with a Spot CCD camera (Invisitron, Puchheim, Germany). Two rows of non-overlapping photographs (~ 80 on average) covering the entire extension of the coverslips were taken from each cell culture for subsequent analysis. Live/Dead assessment was performed by manual counting of DAPI stained nuclei by an observer who was blinded with respect to the culture treatment. Cell counting was supported by using ImageJ Cell counter plug-in (Fiji ImageJ by NIH) as described elsewhere [46 (link)–48 (link)].