Cflow software version 1

Cell labeling of lung and blood leukocytes with monoclonal antibodies was performed as previously described [10 (link),35 (link)]. Separated lung cells or blood leukocytes (1 × 10⁶/well in 100 µL staining buffer) were incubated for 20 min at 4 °C with the following combinations of unlabeled primary monoclonal antibodies to cell surface antigens of camel leukocytes (mAbs; Table 1): CD45/CD172a, CD163/MHC-class II, or CD4/WC1 or with a combination of anti-CD11a-PE and anti-CD18-FITC mAbs. After two washing steps (by adding 150 µL washing buffer followed by centrifugation at 300× g for 3 min), the cells were stained with fluorochrome-labeled antibodies to mouse IgG1, IgG2a, and IgM isotypes for 20 min at 4 °C. In a third staining step, PerCP-labeled monoclonal antibodies to CD14 were added to the cells stained with CD45 and CD172a. Staining with mouse isotype control antibodies was also performed. After the final cell wash, the Acuri C6 flow cytometer (Accuri C6, Becton Dickinson Biosciences) was used for the acquisition of at least 100,000 total cells. CFlow Software, Version 1.0.264.21 (BD Biosciences) was used for data analysis.

The immunophenotype of blood leukocytes was investigated using indirect membrane immunofluorescence and flow cytometry [30 (link)]. In the first labeling step, 4 × 10⁵ leukocytes were incubated with unlabeled primary monoclonal antibodies (mAbs) specific for the cell surface markers, CD4, WC-1, CD172a, CD14, CD163, MHCII, CD44, CD45, CD11a, and CD18 [6 (link)]. After 15 min at 4 °C, the cells were washed twice with MIF buffer and resuspended for the second staining step. After that, the cells were incubated with fluorochrome-labeled secondary antibodies against mouse IgM, IgG1, and IgG2a (Invitrogen). For isotype control staining, class-matching control antibodies (Becton Dickinson Biosciences) were also included. After washing with MIF buffer, labeled cells were analyzed using an Accurie C6 flow cytometer (BD Biosciences) by collecting 100,000 total leukocytes from each sample. Collected data were analyzed with the CFlow Software, Version 1.0.264.21 (BD Biosciences). The total leukocyte number was counted under microscope using the Neubauer counting chamber after staining with Türk solution. The relative fractions of leukocyte subsets as determined by flow cytometry (Figure 1A) were used to calculate their absolute cell numbers.

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).