Cd273 apc

Lymphocytes were isolated as described and stained with anti-mouse IgD PerCP-Cy5.5 (1:400, 405710), LPAM (integrin α4β7) PE (1:100, 120605; (Biolegend, San Diego, CA), B220 V500 (1:400, 561227), CD19 APC-H7 (1:200, 560143), CD80 PE (1:500, 553769), CD273 APC (1:200, 560086), CD138 PE (1:200, 553714), IgM PE-Cy7 (1:200, 552867; BD Biosciences), CCR9 PE-Cy7 (1:100, 25-1991), CD73 PE-Cy7 (1:50, 25-0731), IgA PE (1:50, 12-4204), GL7 eFluor 450 (1:100, 48-5902), CD38 Alexa700 (1:800, 56-0381), CD21/35 Pacific Blue (1:800, 57-0212; eBiosciences) or CCR10 APC (1:100, FAB2815A; R and D systems. Minneapolis, MN) and were analysed using LSR II (BD Biosciences) or Navios (Beckman Coulter, Brea, CA) flow cytometers. For sorting, cells were labelled with anti-mouse CD138 PE (1:200, 553714), CD19 PE-Cy7 (1:200, 552854), CD80 APC (1:200, 560016) and GL7 FITC (1:100, 553666) before sorting using a FACSAria (BD Biosciences). Cells were sorted into tubes that had been coated with 2% BSA/PBS overnight, and pelleted by centrifugation at 600 g before being resuspended in PBS and injected into recipient mice or used for gene sequence analysis.

For phenotypic analysis BM-MSCs, UCT-MSCs and AT-MSCs were harvested and stained for 15 minutes at room temperature with the following monoclonal antibodies (mAbs): anti-human CD90 APC, CD73 PE, CD34 PE, CD200 PE, CD273 APC, CD274 PE, CD71 FITC, CD44 PE (BD Pharmingen, Heidelberg, Germany), anti-human HLA-DR PE, HLA-A,B,C FITC, CD144 (VE-cadherin) APC, CD31 FITC, CD105 PE (Biolegend, San Diego, CA, USA), CD45 FITC (Tonbo, Biosciences, San Diego, CA, USA) or with the appropriate fluorochrome-conjugated isotype-matched mAbs to establish background fluorescence. After incubation cells were washed with PBS, centrifuged at 1400 RPM for 5 minutes and suspended in PBS for flow-cytometry analysis. Samples were acquired using a FACSCalibur (Becton Dickinson, San Josè, CA, USA) and the data were analysed with CellQuest software (Becton Dickinson).