Camel Leukocyte Antigen Profiling by Flow Cytometry

Lymph node cells, blood MNC, or total leukocytes were labeled with monoclonal antibodies (mAbs) to camel leukocyte antigens and analyzed by flow cytometry (13 (link)). Separated cells (1 × 10⁶ cells/well) were incubated in the wells of a 96-well plate with mAbs cross-reactive with the homologous camel molecules: CD45, CD44, BAQ44A, WC1, CD4, CD18, CD172a, CD14, CD163, and MHCII (15 (link), 16 (link)). After incubation (15 min; 4°C), cells were washed twice and were incubated with mouse secondary antibodies (IgM, IgG1, IgG2a; Invitrogen) labelled with different fluorochromes or with mouse isotype control antibodies (Becton Dickinson Biosciences). After washing, cells were analyzed on a Becton Dickinson Accuri C6 flow cytometer (Becton Dickinson Biosciences, San Jose, California, United States). Data of at least 100,000 cells were collected and analyzed with the flow cytometric software C-Flow (Becton Dickinson Biosciences, San Jose, California, United States). For multi-color staining tubes, correction of fluorescence spillover was done after calculation of compensation matrix using the Accuri C6 compensation calculator Excel sheet. For this, a set of single-stained control samples was used to determine the extent of fluorescence spillover from each fluorochrome.