Anti cd11b pe clone icrf44

Cryo-recovered PBMC were cultured at 2.5×10⁶ cells/mL in presence of 0.1 μg/mL LPS (eBioscience), 1 μg/mL of the synthetic toll-like receptor agonist CL097 (In vivogen), or no stimuli in round-bottom 96-well microplates at 37°C and 5% CO₂ for five hours. Two patients with cirrhosis with insufficient cell numbers after cryo-recovery were not stimulated with LPS. After stimulation, supernatants were collected and cells were stained with a panel of antibodies comprising anti-CD1c (clone L161; BioLegend), anti-CD11b-PE (clone ICRF44; BD Biosciences), anti-CD16-BV605 (clone 3G8; BD Biosciences), anti-CD14-APC-Cy7 (clone MφP-9; BD Biosciences), anti-CD141-APC (clone 1A4; BD Biosciences), anti-CD33-PE-Cy7 (clone P67.6; BD Biosciences), anti-CD86-BV711 (clone 2331; BD Biosciences), anti-HLA-DR-BV786 (clone G46-6; BD Biosciences), anti-PD-L1-BUV395 (clone MIHI; BD Biosciences), anti-CD19-PerCP-Cy5.5 (clone SJ25C1; BD Biosciences), and Live/Dead™ Yellow (Life Technologies). Flow cytometric analysis was performed on a five-laser BD LSRFortessa (BD Biosciences). Gatings of cell populations were set based on an internal PBMC control that was run in each experiment.

Fresh BM samples were diluted 1:5 with PBS and analyzed with a hematocytometer to count white blood cells (WBCs), red blood cells (RBCs), and the percentage of neutrophils. Immunophenotyping was then performed using two calibrated mixtures of conjugated mAbs: the first evaluated the myeloid compartment, containing the mAbs anti-HLA-DR-FITC (clone G46-6, BD Biosciences, San Jose, CA, USA), anti-CD11b-PE (clone ICRF44, BD Bioscience), anti-CD45-PerCP (clone 2D1, BD Bioscience, San Jose, CA, USA), anti-CD14-PE-Cy7 (clone M5E2, BD Bioscience), anti-CD33-APC (clone WM53, BD Bioscience), and anti-CD16⁻ APC-Cy7 (clone 3G8, BD Bioscience), and the second evaluated the lymphoid compartment, composed of the Abs anti-CD56-PE (clone MY31, BD Bioscience), anti-CD8-PE-Cy7 (clone RPA-T8, BD Bioscience), anti-CD3-APC (clone SK7, BD), anti-CD19-APC-Cy7 (clone SJ25C1, BD Bioscience), and anti-CD4-FITC (clone L200, BD Bioscience). Cells were stained for 30 min at 4 °C in the dark in PBS containing 2% FCS buffer, washed, and analyzed using a Navios Flow Cytometer (Beckman Coulter Inc. Brea, CA, USA).