Cd8 alexa fluor 700

For flow cytometric analysis of HBEC, cells were incubated in the presence of fluorochrome-conjugated mAbs against CD105 (SN6), VCAM-1 (STA), B7-1/CD80 (2D10.4), B7-2/CD86 (IT2.2), CD40 (5C3), HLA-DR/MHC II (LN3) and CD275 (MIH12) (all from eBioscience), ICAM-1 (5.6E; Beckman Coulter) and β₂-microglobulin/MHC I (TÜ99; BD Biosciences) as per manufacturer’s instructions. Flow cytometric analysis was performed on the FC 500 flow cytometer. For flow multi-colour cytometric analysis of the activation status of donor T cells, cells were incubated with the following antibody cocktail: CD69-FITC (FN50), CD40L-PE (CD154, 24-31) (eBioscience), CD8-Alexa Fluor 700, CD4-Pacific Blue, CD45RA-Alexa Fluor 750, CD3-Krome Orange (Beckman Coulter) and CD62L-APC (Miltenyi Biotec). Cells were washed once prior to flow cytometry using a Gallios™ Flow Cytometer (Beckman Coulter). Naive and memory T-cell populations were distinguished by CD45RA and CD62L expression, as described previously (37 (link)). For EMP phenotyping, HBEC supernatant following stimulation was incubated with mAb and diluted 1:1 in PBS prior to flow cytometric analysis on the FC 500 flow cytometer. EMP production per 100,000 HBEC was determined by triplicate supernatant analysis. The supernatant was collected from 400,000 cultured HBEC per well following 18 h stimulation.