Cd16 cd32 blocking antibodies

Normal brains of adult Ang-2 GOF mice and wild-type littermates were dissected and immediately transferred in ice-cold HBSS. Briefly, following gentle mincing, the tissue was incubated in an HBSS solution containing Collagenase P (0.2 mg/ml), Dispase II (0.8 mg/ml), DNase I (0.01 mg/ml), Collagenase A (0.3 mg/ml) for 60 min at 37 °C under gentle rocking as previously described [52 (link)]. Following myelin removal, the cells were preincubated with rat anti-mouse FcγIII/II receptor (CD16/CD32) blocking antibodies (≤1 μg/million cells/100 μl; BD) for 5 min at 4 °C, and stained with the fluorochrome-conjugated antibodies (0.25–1 µg): CD45 PercP Cy5.5 (clone 30-F11, BD Biosciences), Gr-1 APC-Cy7 (clone RB6-8C5, BD Biosciences), CD11b APC (clone M1/70, BD), F4/80 FITC (clone BM8, eBioscence). Following two washing steps, DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added for live gating and cells were acquired on a FACSCanto™ II flow cytometer (BD) using Diva Software (BD) and further analyzed using FlowJo analytical software (FlowJo Version 10.0.8, LLC). Background fluorescence levels were determined by Fluorescence Minus One (FMO).

Mice subjected to spinal cord injury were killed by an overdose of anesthetic and their spinal cords, processed for flow cytometry after saline transcardial perfusion. The lesioned cord segments extending between T11 and T13 were homogenized and reduced to single-cell suspensions by Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) according to the manufacturer’s instructions. Cell suspension was filtered through a 70-μm strainer, centrifuged over a 30% discontinuous Percoll Gradient (GE Healthcare) and myelin debris removed from the surface (modified from Cardona et al., [28 (link)]). Cells were labeled with Zombie Aqua™ die (Biolegend), then incubated with 5% FBS, 1%BSA, and 5 mg/ml rat anti-mouse Fc III/II receptor (CD16/CD32) blocking antibodies (BD), and then stained using the following monoclonal antibodies: CD45-Pb (Biolegend), CD11b-PeCy7 (BD), Ly6C-APCCy7 (BD), Ly6G-PerCP Cy5.5 (BD), and CD11c-APC (ebioscence). Cells were analyzed by LSR Fortessa (Beckton Dickinson) and data analyses by FlowJo (Treestar) software.