Quantifying Cell Survival from Microscopy

Both hemocyte and neuron survival was analyzed as described previously (Miljus et al., 2014 (link); Hahn et al., 2017 (link), 2019 (link)). After fixation, coverslips with attached cells were washed (5 min per step) three times in PBS followed by two wash steps in PBS/0,1% Triton-X-100 (PBST). Cells were stained with Dapi (1:1000 in PBST; Sigma-Aldrich; Munich, Germany) for 30 min in the dark. Subsequently, coverslips were washed five times in PBS before mounting on microscopy slides in DABCO (Roth, Karlsruhe, Germany).
Coverslips were imaged with an epifluorescence microscope (Zeiss Axioskop; Oberkochen, Germany; 40x objective was used for locust neurons or hemocytes, 63x oil objective was used for tribolium neurons) equipped with a Spot CCD camera (Invisitron, Puchheim, Germany). Non-overlapping series of photographs passing the center of the coverslip to the left and the right were taken from all cultures (~80 pictures per locust culture and ~120 pictures per tribolium culture). Cells were manually scored as intact or dead/dying on the basis of Dapi-fluorescence pattern reflecting nuclear chromatin structure. The scorer was blinded with respect to the culture treatment during counting. Cell counting was supported by ImageJ Cell counter plug-in (Fiji ImageJ by NIH) as described elsewhere (Miljus et al., 2014 (link); Hahn et al., 2019 (link); Knorr et al., 2020 (link)).