4.7. Immunofluorescence Analysis

For immunofluorescence analysis, iPSCs were cultivated in 24 well plates and fixed with 2% (w/v) paraformaldehyde. Staining of surface expressed DAF and CAR was carried out without permeabilization, whereas F-actin staining with Alexa Fluor 555 phalloidin (Thermo Fisher Scientific) at a 1:40 dilution was applied after permeabilization with 0.3% Triton X-100 in PBS. After blocking of unspecific binding with goat or donkey serum, anti-CAR (#sc-15405 H-300, Santa Cruz Biotechnology or clone RmcB) or anti-DAF (#sc-133220 H-7, Santa Cruz Biotechnology) antibodies were applied at a dilution of 1:100 followed by addition of goat anti-rabbit or donkey anti-mouse IgG (H + L) Cy3 MinX secondary antibodies (Dianova, Hamburg, Germany). Addition of primary and secondary antibodies and phalloidin dye was followed by application of the DNA-specific stain Hoechst bisbenzimide 33258 (Thermo Fisher Scientific) at 5 µg/mL in PBS. Immunofluorescence analysis of CAR with clone H-300 was done in reference to our previous publication [23], which showed differential surface expression of CAR on TMOi001-A and WISCi004-A iPSC. This was complemented by the application of the well-characterized RmcB clone, which was also used for antibody blocking experiments. Supplementary Figure S2 shows a representative flowcytometric analysis on the lack of fluorescence signal after application of the donkey anti-mouse Cy3 secondary antibody. Images were taken with an Olympus XM10 fluorescence microscope and processed for minimal alterations in contrast and background in CorelDRAW2020.