Cxcr3 pe cy7

Immunophenotypic analyses of splenoctyes and single cell suspensions from tumors were assessed by flow cytometry. Antibodies to stain for MDSC were CD11b-APC (Clone M1/70; BD Biosciences), Ly6G-FITC (Clone 1A8; BD Biosciences), Ly6C-PE (Clone AL-21; BD Biosciences). For T cell activation markers, cells were stained with Ab specific for CD4-PE-Cy7 (Clone RM4-5; BD Biosciences), CD8-PE-Cy7 (Clone 53-6.7; BD Biosciences), CD62L-PE (Clone MEL-14; BD Biosciences), and CD44-FITC (Clone IM7; Biolegend). To determine Th1 and Th2 phenotypes, cells were stained using fluorochrome-conjugated Ab targeted CXCR3-PE-Cy7 (Clone CXCR13-173; Biolegend), CCR4-PE (Clone 2G12; Biolegend), and CCR6-APC (Clone CK4-L3; BD Biosciences). Cells were incubated on ice for 30 minutes, washed, and fixed in PBS containing 1% formalin for flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).

Approximately 1 x 10⁶ T cells were washed with cold FACS staining buffer (1x PBS with 2% FBS) and stained with either: PE-conjugated control unloaded (empty) mouse MR1(K43A) tetramer or PE-conjugated rRL-6HM-loaded mouse MR1(K43A) tetramer at 20 μg/ml (1G tetramers; (4 (link))); or PE-conjugated non-stimulatory control mouse MR1(wild type)–6-FP tetramer or PE-conjugated mouse MR1(wild type)–5-OP-RU tetramer at 1.4 μg/ml (2G tetramers; (6 (link))) for 45 minutes at room temperature in the dark. Immediately thereafter, cells were co-stained for 30 minutes on ice with mixture of selected fluorochrome-conjugated antibodies including anti-mouse CD3-Pacific blue (BD Biosciences), CD4-PerCP or Alexa Fluor 700 (BioLegend), CD8α-APC-H7 (BD Biosciences), CD8β-Alexa Fluor 647 or PerCP-Cy5.5 (BioLegend), NK1.1-PerCP-Cy5.5 or -BV510 (clone PK136, BD Biosciences), CD69-APC, CD44-PE-Cy7, Vβ6/Vβ8.1–8.2 TCR-FITC (BD Biosciences), CXCR3-PE-Cy7 and α4β1 integrin-Alexa Fluor 647 (BioLegend). After washing once with 2 ml of FACS staining buffer, data was acquired on BD FACS CantoII, BD LSR II or BD FACSAria flow cytometers and analyzed using FlowJo analysis software (Tree Star).