Anti cd16 cd32 mab

The freshly harvested cells were analyzed by flow cytometry essentially as described [27 (link)]. Briefly, red blood cells were removed following centrifugation by re-suspending the harvested cells in the 1X red blood cell lysis buffer for 5 min. The cells were centrifuged again, washed 3 X with PBS buffer, and the cell number was determined with Beckman-Coulter Z1 Automated Cell Counter. Cell viability was assessed using trypan blue exclusion and cell morphology was examined using light microscopy. For all experiments, cell suspensions were preincubated with anti-CD16/CD32 mAb (Biolegend) to block FcγRII/III receptors. Cell staining was performed in the dark for 30 min at 4°C in staining buffer. The reagents used for flow cytometry and the list of antibodies were shown in S1 Table and S2 Table, respectively. Cells were analyzed on Cedars-Sinai Flow Cytometry Core Facility instruments (Beckman-Coulter (Dako, CyAn). Data were analyzed with Summit software. The engrafted GFP⁺ cells were re-gated for the expression of CD11b/F4/80 (monocytes/macrophages), Siglec-F (eosinophils), and Ly6G (neutrophils). For more information about the flow cytometry reagents see S1 Table and S2 Table.

J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 µg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA. After washing three times with PBS (−), the cells were stained with streptavidin-Alexa Fluor 568 conjugate (1:1,000 dilution, Invitrogen) and anti-mouse IgG-Alexa Fluor 488 conjugate (1:1,000 dilution, Invitrogen) at 4 °C for 15 min in HBSS-BSA. After washing, the stained cells were fixed with 4% paraformaldehyde in PBS (−) for 10 min at room temperature and further stained with 1 µg/ml 4’,6-Diamidino-2-phenylindole (DAPI, Dojindo). Fluorescent images were obtained using a confocal microscope (FV1200, Olympus) and analyzed by Olympus software (FV10-ASW, Olympus).