Alexa flour 647

Platelet activation was quantitated by surface expression of CD62P and activated integrin α_IIbβ₃ following stimulation with either 0.5 units/ml thrombin for 5 min or PCa cells at the indicated cell:platelet ratios for 24 h. Briefly, 100 μl of activated platelet suspensions were fixed with 2% paraformaldehyde (PFA) and subsequently stained with an Alexa Flour 647 conjugated antibody recognizing CD61 (BioLegend, San Diego, CA, Cat #336407, RRID: AB_2128751) and a fluorescein isothiocyanate (FITC) conjugated antibody specific for CD62P (P-selectin antibody; BD Biosciences, San Jose, CA, Cat #555523, RRID: AB_395909), or stained directly (no fixative) with a FITC conjugated antibody recognizing PAC-1 (activated α_IIbβ₃; BD Biosciences, San Jose, CA, Cat #340507, RRID: AB_2230769). Platelets were initially gated on the expression of platelet marker CD61 and then subsequently analyzed for expression of CD62P or activated α_IIbβ₃ by flow cytometry (Accuri C6 Plus, BD Biosciences, San Jose, CA).

Cells were fixed with 2% paraformaldehyde (PFA) w/o methanol (15 min, Thermo Fisher Scientific, Darmstadt, Germany), permeabilized, and blocked with 0.5% Triton X-100 (Thermo Fisher Scientific, Waltham, USA) in 2% BSA (PAN-Biotech, Aidenbach, Germany) for 60 min. ER stress markers included: Alexa 647 anti-ATF-4 antibody (B-3, 1:50, Santa Cruz), Alexa 594 anti-calnexin antibody (AF18, 1:50, Santa Cruz), and Alexa 488 anti-cytochrome c (1:50, Biolegend). For stemness, antibody mixtures, either containing anti-GFAP (1:200, Alexa Fluor 594, BioLegend, San Diego, USA) and anti-A2B5 (1:200, Alexa Flour 647, BioLegend, San Diego, USA), or anti-Oct-4 (1:500, Alexa Fluor 647, BioLegend, San Diego, USA) and anti-Nanog (1:500, Alexa Flour 488, BioLegend, San Diego, USA) were added and staining was done at 4 °C overnight. The next day, GFAP/A2B5-antibody mix was stained with Phalloidin green (1:50, BioLegend, San Diego, USA). Nuclei were counterstained with DAPI (1:1.000, Biomol, Hamburg, Germany) and cells were analyzed using a Zeiss microscope Axio Observer 7 (Zeiss, Oberkochen, Germany).