Anti mouse igg1 pe

T cells were stained with primary antibodies against HSP90α (2G5.G3, Abcam, USA), HSP90β (EPR16621, Abcam, USA), HSP70 (C92F3A-5, Enzo Life Sciences, USA), HSC70 (1B5, Stressgen, USA), HSP40/DNAJB1 (3B9.E6, Abcam, USA), GRP78 (StressMarq Biosciences, USA), and HSP60 (StressMarq Biosciences, USA). Secondary antibodies were goat anti-rabbit IgG H&L (PE) preadsorbed (Abcam), anti-mouse IgG1 (PE) (Miltenyi Biotec, Germany), and anti-rat F(ab’)2 (PE) (Sony Biotechnology, USA). T cells were also stained with anti-human CD279/PD-1 (REA1165, Miltenyi Biotec, Germany) (PE), CD152/CTLA-4 (REA1003, Miltenyi Biotec, Germany) (PE), primary antibody against STAT3 (4G4B45, Sony Biotechnology, USA), and secondary antibody anti-mouse IgG1 (PE) (Miltenyi Biotec, Germany). The FcR blocking reagent (Miltenyi Biotec, Germany) and isotype controls were used to exclude non-specific binding. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm. Cells were analyzed by flow cytometry.

Culture organoids were harvested using cold PBS and dissociated into single cells by incubation with TrypLE Express. Single cell suspensions were washed with FACS buffer (0.5 % BSA in 1 X PBS) and stained with anti-CD326 (APC/human; Miltenyi, Germany), anti-CD326 (APC/mouse; Miltenyi, Germany), anti-CD44 (APC/mouse; Miltenyi, Germany) and anti-rat IgG2b (APC; Miltenyi, Germany), anti-mouse IgG1 (PE; Miltenyi, Germany), anti-human IgG1 (APC; Miltenyi, Germany), anti-human IgG1 (PE; Miltenyi, Germany) for 30 min. Cells were washed 3 times and finally resuspended in 400 μl of FACS buffer. All samples were run on a Cyto-FLEX-Analyzer (Beckman coulter, USA) and data were analyzed by CytExpert software (Beckman coulter, USA).