Cd8α apc h7

Prior to perfusion, spleens were harvested from experimental mice. Single cell suspensions from spleens were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25 mL of ice-cold PBS and brains and spinal cords were collected from perfused mice and dounce homogenized to obtain single cell suspensions. CNS cells were purified by centrifugation for 30 min in a 30% Percoll (GE Healthcare) solution as previously reported (25 (link)). Cells were incubated with the anti-Fc receptor antibody 2.4G2 prior to the addition of antibodies. The following antibodies were purchased from BD Biosciences: CD45-FITC, CD8α-APC-H7, CD19-APC-H7, CD19-BV510, B220-PE-TxRed, B220–PE-CF594, CD11b-AlexaFluor-700, MHC-v450. The following antibodies were purchased from eBioscience: MHCII-Pacific Blue, CD11c-PECy7. The following antibodies were purchased from BioLegend (San Diego, CA): CD138-PE, MHCII (I-A/I-E)-Pacific Blue, Thy1.1-PerCP, CD4-APC. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed with FloJo software (TreeStar) with doublets being excluded.

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).