Anti cd4 fitc

Primary mouse antibodies used for western blotting and immunostaining were rat anti-K8 and rat anti-K19 (Troma I respectively Troma III, Hybridoma bank, Iowa, USA), mouse anti-K7 (RCK 105, Abcam, Cambridge, UK), mouse anti-K20 (IT-Ks 20.10, Progen, Frankfurt, De), rat anti-K18 (Troma II, Hybridoma bank, Iowa, USA) [24 (link)], mouse anti-tubulin (Sigma, Munich, Germany), rabbit anti-caspase-7 and anti-cleaved caspase-7 (Cell Signaling, Danvers, MA, USA), rabbit anti-MPO (Thermo Scientific, Waltham, MA, USA) and rat anti-Hsc70 (Stressgen, Victoria, Canada). The secondary antibodies used for staining were Alexa 488 or Alexa 546 anti-mouse, Alexa 488 anti-rat and Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). The secondary antibodies used for western blotting were: anti-mouse HRP (GE Healthcare, Little Chalfont, UK), anti-rabbit HRP (Cell Signaling, Danvers, MA, USA) and anti-rat HRP (GE Healthcare, Little Chalfont, UK) antibodies. Nuclei were stained with Draq5 (Cell Signaling, Danvers, MA, USA) or Dapi (Invitrogen Carlsbad, CA, USA). Antibodies used for FACS analysis were anti-CD4-FITC and anti-CD49d-PE or anti-L-selectin-PE (Immunotools, Friesoythe, Germany).

ES-2 and ES-2.PDL1KO cells were suspended in 100 µL of PBS, stained (30 min, 4 °C) with anti-PDL1-APC (Biolegend) or mouse IgG2a-APC (Biolegend) as isotype control, washed with PBS and analyzed by flow cytometry. After 4 days of coculture with ES-2scFv:CD3-mcherry cells, CD3⁺ T-cells were furthermore collected and stained for CD4 (anti-CD4-FITC; Immunotools), CD8 (anti-CD8-Pacific Blue; Beckman Coulter), CD25 (mouse anti-CD25-APC; Immunotools), mouse anti-41BB-APC (Immunotools) and with the zombie NIR fixable viability kit (Biolegend).