Accurie c6 flow cytometer

ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) [32 (link)]. Stimulated and non-stimulated leukocytes (1 × 10⁶/well) were incubated for 20 min (37 °C, 5 % CO₂) with the ROS-sensitive dye dihydrorhodamine (DHR)-123, (750 ng/ml final, Mobitec, Goettingen, Germany). After incubation, cells were washed with MIF buffer, and the percentage of ROS-positive cells and the relative amount of generated ROS was determined by flow cytometry (Accurie C6 flow cytometer, BD Biosciences) after acquisition of 100 000 events (Fig. 2A-B).

Cell labeling was performed in a round-bottomed 96-well microtiter plate using 5 × 10⁵ leukocytes per well as previously described [38 (link)]. All incubation and centrifugation steps were performed at 4 °C. Separated leukocytes were incubated with unlabeled primary monoclonal antibodies (mAbs) (Supplementary Table S1) specific for the cell surface molecules CD4, WC-1, CD14, CD163, CD172a, MHC-II, CD11a, CD18, CD44, and CD45R [15 (link),34 ,35 (link),36 (link),37 (link)] for 15 min in the dark. After two washings in staining buffer, the cells were incubated with fluorochrome-labeled anti-mouse IgM, IgG1, and IgG2a secondary antibodies (Invitrogen, Schwerte, Germany) for 15 min in the dark. Parallel setups were incubated only with antibody isotype controls. After two washings, labeled cells were analyzed on an Accurie C6 flow cytometer (BD Biosciences, Heidelberg, Germany) by the acquisition of at least 100,000 total leukocytes. Collected flow cytometric data were analyzed using the CFlow Software (V 1.0.264.21; BD Biosciences, Heidelberg, Germany). Leukocyte count was estimated under a microscope using the Neubauer counting chamber after staining of the blood sample with Türk Solution.

The expression levels of several cell markers were analyzed using membrane immune cell labeling and flow cytometry [24 (link)]. Isolated camel blood leukocytes were suspended in PBS supplemented with bovine serum albumin (5 g/L) and NaN₃ (0.1 g/L) (MIF buffer; membrane immunofluorescence buffer) at 5 × 10⁶ cells/mL. Leukocyte suspension (100 µL containing 4 × 10⁵ cells) was incubated with primary monoclonal antibodies (mAbs) against the following cluster of differentiation (CD) antigens: CD4, WC-1, CD14, CD163, and major histocompatibility complex (MHC) class II molecules [25 (link)]. To remove the unbound antibodies, the cells were washed twice with MIF buffer (by addition of 150 µL buffer followed by centrifugation for 3 min at 300× g and 4 °C). To detect the cells labeled with mouse primary antibodies, fluorochrome-conjugated secondary antibodies directed against mouse isotypes (IgM, IgG1, IgG2a; Invitrogen; Schwerte, Germany) were added to the cells. Control set-ups were stained with isotype control antibodies. Finally, labeled cells were washed twice with MIF buffer and analyzed using flow cytometry (Accurie C6 flow cytometer, BD Biosciences). After the measurement of 100.000 total leukocytes, data were analyzed using the CFlow Software (BD Biosciences; Heidelberg, Germany).

ROS generation was performed in 96-well, round-bottom microtiter plates (Corning, NY, USA) [10 (link)]. Leukocytes (1×10⁶/well) were incubated for 30 min (37°C, 5% CO₂) with heat-killed S. aureus bacteria (30 bacteria/cell) in the presence of ROS-sensitive dye dihydrorhodamine (DHR)-123 (750 ng/mL final; Mobitec, Goettingen, Germany). For monocyte identification, the cells were incubated with APC-conjugated monoclonal antibodies specific to CD14. After incubation, the cells were washed with PBS, and the percentage of ROS-positive cells and the relative amount of generated ROS were determined by flow cytometry (Accurie C6 Flow Cytometer; BD Biosciences, USA) after the acquisition of 100,000 events.

For the analysis of changes in neutrophil side scatter characteristic (SSC), which is indicative of neutrophil degranulation and secretion of their granular contents [32 (link),33 (link)], separated leukocytes (5 × 10⁶ cells/mL) were analyzed for their mean SSC values using the Accurie C6 flow cytometer (equipped with a blue (488 nm) and a red (640 nm) laser, two light scatter detectors (FSC and SSC), and four fluorescence detectors; BD Biosciences, Heidelberg, Germany). Neutrophil SSC height (SSC-H) was measured using the CFlow^® software (version 1.0.264.21, BD Biosciences, Heidelberg, Germany) and compared between cells from normal range animals and animals during restricted housing.