Optilyse c lysis solution

Percoll media (GE Healthcare, Bio-Sciences AB) of different densities were prepared as previously described [17 (link)]. A BM cell suspension or whole blood (3 mL) was gently layered on top of a two-layer discontinuous Percoll gradient: 1.093 and 1.077 g/mL (72 % and 60 % Percoll) densities for BM; 1.068 and 1.058 g/mL (51 % and 42 % Percoll) densities for blood. The tubes were then centrifuged at (1000 × g, 20 min, room temperature; Multifuge 1R, Thermo Fisher, USA) to separate the rat BM into three fractions. The middle-level cell fraction, which contained PMNLs, was washed twice in 10 mL of PBS (300 × g, 10 min, room temperature) to remove the remaining Percoll, and erythrocytes contaminating the pellet were removed using an Optilyse C lysis solution (Beckman Coulter, France). The PMNLs, separated by Percoll, were confirmed as BM neutrophil cell lines at various stages of differentiation by flow cytometry and microscopy using oil lens [18 (link)]. We used only samples containing >90 % BM PMNLs (bands and segmented granulocytes) and circulating PMNLs with 95 % cell viability, as determined by trypan blue staining.

To quantify CLEC5A expression levels in granulates and monocytes, 1 ml samples of whole blood were collected and stained with phycoerythrin- (PE-) conjugated anti-CLEC5A monoclonal antibody (mAb) (R&D Systems, Minneapolis, MN, USA) and phycoerythrin-cyanin 5- (PC5-) conjugated anti-CD14 mAb (Beckman Coulter, Brea, CA, USA) or fluorescein isothiocyanate- (FITC-) conjugated CD66b-specific mAb (Beckman Coulter, Brea, CA, USA) according to the manufacturer's protocol and the described technique [12 (link)]. Mouse IgG2b-PE (R&D Systems, Minneapolis, MN, USA) and IgG2a-PC5 (Beckman Coulter, Brea, CA, USA) were used as isotype controls. Samples were incubated with antibodies for 20 minutes in the dark at room temperature, and then, erythrocytes were lysed by 500 μl of OptiLyse C Lysis Solution (Beckman Coulter, Brea, CA, USA) for 10 minutes to lyse red blood cells. Then, the cells were resuspended with 500 μl PBS/each tube prior to flow cytometry (Beckman Coulter, Brea, CA, USA) analysis. Monocytes and granulocytes were gated based on CD45+/side scatters (SSC) and at least 2 × 10⁵ total cells from each sample were analyzed. Data were expressed as the percentages or the mean fluorescence intensity (MFI) of CLEC5A expression in circulating monocytes or granulocytes.

To quantify expression levels of MDL-1, 1000 µl samples of whole blood were obtained and stained with phycoerythrin (PE)-conjugated anti-MDL-1 mAb (R&D Systems, Minneapolis, MN, USA) and Phycoerythrin-Cyanin 5 (PC5)-conjugated anti-CD14 mAb (Beckman Coulter, Brea, CA, USA) according to protocols of the respective manufacturers. Fluorescent antibodies, mouse IgG2b-PE (R&D Systems, Minneapolis, MN, USA) and IgG2a-PC5 (Beckman Coulter, Brea, CA, USA) were used as isotype controls. After incubation for 20 minutes in the dark at room temperature, cells were reacted with 500 µl of OptiLyse C Lysis Solution (Beckman Coulter, USA) for 10 minutes to lyse red blood cells. Then, 500 µl PBS was added into each tube to stop the reaction prior to flow cytometry (Beckman Coulter, Brea, CA, USA) analysis. Monocytes were gated on the basis of CD14+/side scatters (SSC) and at least 2 x10⁵ total cells from each sample were analyzed.