αcd8α percp cy5 5 clone 53 6

Splenocytes were stimulated for 6 hours with DMSO or GUCY2C_254–262 and Protein Transport Inhibitor Cocktail (eBioscience). Cells were stained with Live/Dead fixable Aqua Dead Cell stain kit (Invitrogen) and αCD8α-PerCP-Cy5.5 (clone 53-6.7, BD Pharmingen) and ICS staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences) and the following antibodies: αIFNγ-APC-Cy7 (XMG1.2) and αTNFα-PE-Cy7 (MP6-XT22) from BD Biosciences, and αMIP1α-PE (clone 39624, R&D Systems). Cells were fixed in 2% PFA and analyzed on a BD LSR II flow cytometer. Analyses were performed using FlowJo software (Tree Star).

TCR-transduced T cells were stimulated for 6 h with DMSO, GUCY2C_153–167 or OVA_323–339 peptides, or with Cell Stimulation Cocktail (PMA/Ionomycin, Thermo Fisher Scientific, Waltham, MA). Incubation included the Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific) when assessing intracellular cytokines. Transduced T cells or blood obtained by retro-orbital eye bleeding were stained for surface markers using the following antibodies: αCD4-Pacific Blue (clone RM4–5, BD Biosciences), αCD8α-PerCP-Cy5.5 (clone 53.6–7, BD Biosciences), αGr1-APC (clone RB6–8C5, BioLegend, San Diego, CA), αB220-PerCP-Cy5.5 (clone RA3–6B2, BD Biosciences), αTer119-PE-Cy7 (clone TER-119, BD Biosciences), and αCD45.2-PE (clone 104, BD Biosciences) and αCD25-PE (clone PC61.5, eBioscience). ICS was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences) and staining with the following antibodies: αGFP-Alexa-488 (polyclonal, Invitrogen) and αIFNγ-PE-CF594 (clone XMG1.2), αIL-2-APC (clone JES6–5H4) and αTNFα-PE-Cy7 (clone MP6-XT22) from BD Biosciences. Red blood cells were lysed using BD lysis buffer and cells were fixed in 2% PFA and analyzed on a BD LSR II or BD LSR Fortessa flow cytometer. Analyses were performed using FlowJo software (FlowJo, Ashland, Oregon).