Anti cd11b fitc

Monocyte subset analysis was performed using FACS Calibur flow cytometer (Becton Dickinson, United States) with monoclonal antibodies: anti-CD14-FITC, anti-CD14-PE, anti-CD11b-FITC, anti-CD18-PE, anti-CD54-FITC, anti-CD36-FITC, anti-CD16-PE, anti-CD206-PE (IO test, Beckman Coulter, France). 50 μl EDTA whole blood was stained with 5 μl of the relevant antibodies for 20 min at room temperature. In order to reduce Fc-receptor mediated binding of test antibodies, 2.5 μl Human Seroblock reagent (BIO-RAD, United States) were added for 10 min at room temperature. Red blood cells were lysed with 250 μl OptiLyse C Lysing Solution (Beckman Coulter, France) before analysis.

Leukocytes in whole blood were fixed with 0.2% paraformaldehyde and the erythrocytes lysed by ammonium chloride according to Hamblin et al.^{32 (link)}
After two washes with PBS containing 0.1% human albumin [PBS–0.1% human serum albumin (HSA)] the leukocytes were incubated for 30 min at +4°C in two tubes with fluorescence labelled antibodies. Tube 1 contained anti-CD11b-FITC [Beckman Coulter (BC) Marseilles, France], anti-CD14-PC7 (BC), anti-CD15s-BV421 (BD Biosciences, San Jose, CA, USA) and anti-CD66b-APC (BC), and tube 2 anti-CD65s-FITC (Bio-Rad, anti-CD14-PC7 (BC), Oxford, UK), anti-CD64-APC-AlexaFluor750 (BC), and anti-CD162-PE (BD Biosciences). After incubation, all the samples were washed twice with ice cold PBS and thereafter diluted in PBS–0.1% HSA. The tubes were placed in the dark at 4°C until the flow cytometry analysis was performed.

Three milliliters of PBMCs with a concentration of 5 × 10⁶ cells/ml was cultured in a well of a 6-well plate. After 1 h of incubation at 37°C and 5% CO₂, the nonadherent cells were washed 3 times with warm PBS. The remaining adherent monocytes were prestimulated for 24 h with concentrations of different ligands that would induce either training or tolerance, such as β-glucan (1 μg/ml), MDP (1 μg/ml), flagellin (1 μg/ml), R848 (1 μg/ml), Pam3CSK4 (1 μg/ml), and RPMI (negative control). The stimuli were washed away and the cells were further incubated for 5 days in the presence of RPMI supplemented with 10% pooled human serum. On day 7, the cells were incubated for 1 h with Versene solution, collected, harvested by centrifugation, and suspended in PBS supplemented with 1% protein blocking agent (PBA). The cells were washed two times and stained extracellularly with the following antibodies: anti-CD45-PeCy7 (Beckman Coulter), anti-CD68-APC (BioLegend), anti-CD14-PE (Beckman Coulter), anti-CD11b-FITC (Beckman Coulter), and anti-DC-SIGN-APC (BioLegend). The samples were measured on a fluorescence-activated cell sorter (FACS) FC500, and the data were analyzed using the CXP software (Beckman Coulter).