Mouse isotype control antibodies

The expression of different myeloid markers and cell adhesion molecules was analyzed using membrane immunofluorescence and flow cytometry [30 (link)]. Separated leukocytes (4 × 10⁵) were incubated with unlabeled primary monoclonal antibodies (mAbs) specific for the cell markers CD14 and MHCII or with directly labeled monoclonal antibodies to the cell adhesion molecules CD11a and CD18 [31 (link)]. After incubation (15 min; 4 °C), cells were washed twice and cells labeled with anti-CD14 and anti-MHC class II molecules were incubated with mouse secondary antibodies (IgG1, IgG2a; Invitrogen) labeled with different fluorochromes. Mouse isotype control antibodies (Becton Dickinson Biosciences) were also included. Washed cells were analyzed using the Accurie C6 flow cytometer (BD Biosciences). At least 100 000 total leukocytes were collected and analyzed with the CFlow Software, Version 1.0.264.21 (BD Biosciences).

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.

The expression of the monocytes markers CD163 and major histocompatibility complex (MHC) class II molecules as well as the cell adhesion molecules CD11a and CD18 was analyzed using membrane immunofluorescence test as previously described (38 (link)). Separated leukocytes (4 × 10⁵) were incubated (15 min; 4°C) with unlabeled primary monoclonal antibodies (mAbs) specific for the cell markers CD14, CD163, and MHC-class II or with directly labeled mAbs to the cell adhesion molecules CD11a and CD18 (16 (link)). After two washing steps, the cells labeled with primary anti-CD14, anti-CD163, or anti-MHC class II molecules (mAbs) were incubated with mouse secondary antibodies (IgG1, IgG2a; Invitrogen) labeled with different fluorochromes. Mouse isotype control antibodies (Becton Dickinson Biosciences) were also included. Washed cells were analyzed using the Accuri C6 flow cytometer (BD Biosciences). At least 100 000 total leukocytes were collected and analyzed with the CFlow Software, Version 1.0.264.21 (BD Biosciences).