Viastain calcein am

Tumor colon organoids expressing GFP-luc2 were infected and treated as described before. At 48 h of post-infection with VSVΔ51-RFP, GFP and RFP fluorescence was visualized with BioTek Cytation C10 confocal imaging reader (Aglient) at 2.5X magnification. Images were stitched with Gen5 software to have an overview of the whole well in a 48-well plate. GFP/RFP fluorescence areas were quantified using the ImageJ software and the percentage of infectivity was calculated as the ratio of GFP-positive (total organoid area) to RFP-positive (infected) area. Organoids that were not transduced with pGL4.10[luc2] were stained with Calcein green (ViaStain Calcein AM, Nexcelom Bioscience) to quantify the initial GFP-organoid area. To assess the robustness of the method, the percentage of infectivity with VSVΔ51 in sensitive versus resistant to the infection tumor organoids was measured by microscopy and further processed by flow cytometry and compared side-by-side.

To visualize viable cells by fluorescence microscopy staining with acetoxymethyl precursor of calcein (ViaStain Calcein AM, Nexcelom Bioscience) was performed. Calcein, a dye capable of permeating cell membranes, can be introduced into cells through incubation. Following the entry into the cells, endogenous esterase hydrolyzes calcein, transforming it into the intensely negatively charged green fluorescent calcein, which is exclusively retained in the cytoplasm of viable cells. 4× staining solution prepared in PBS was added to the culture media of wells containing cells or organoids and incubated for 30 min in the dark at 37 °C.