Multiparameter Flow Cytometry Analysis of MR1-Restricted T Cells

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).