Apc mouse anti human cd11b

Within three hours from sampling, 100 µL of blood was transferred into 1.5 mL tubes. Labeling of cells was made with the following antibodies from BD Biosciences (Stockholm, Sweden): APC mouse anti-human CD11b, PE mouse anti-human CD87, APC mouse anti-human CD14 and APC mouse-anti human CD3. Corresponding isotype antibodies were used as negative controls. The tubes were incubated at room temperature for 30 min and erythrocytes were lysed using 1 mL FACS Lysing Solution (BD Biosciences, Stockholm, Sweden) for 30 s at room temperature. The cells were then washed with 1 mL PBS (Life Technologies Europe BV, Stockholm, Sweden) with 1% BSA (Sigma-Aldrich Sweden AB, Stockholm, Sweden). The tubes were centrifuged at 1000×g for five minutes and the supernatant was discarded and the washing procedure was repeated once more. Finally, the cells were resuspended in 0.5 mL CellFIX (BD Biosciences, Stockholm, Sweden) and protected from light before flow cytometric analysis.
The flow cytometric analysis was performed on an Accuri C6 (BD Biosciences, Stockholm, Sweden) with the associated software CFlow Plus (BD Biosciences, Stockholm, Sweden). For each sample, data were collected from 25,000 cells in the total leukocyte gate.

Adherent cells were harvested by trypsinization. Cells were suspended in 1% BSA/PBS buffer at a concentration of 10⁵ cells/100 μl. Cell suspensions were incubated with BV711 Mouse Anti-Human CD45 (1:20; cat. #5643257, BD Biosciences, Franklin Lakes, NJ, USA) and APC Mouse Anti-Human CD11b (1:5; cat. #550019, BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4 °C in the dark. Cells were washed 2 times with 1%PBS/BSA, resuspended in 1 ml 1%PBS/BSA and analyzed by BD FACS Aria II Cell Sorter (BD Life Sciences, San Jose, CA, 95131, USA). Unstained cells were used as negative controls.