Cd11b apc cy7 antibody

Microglial phagocytosis was performed similarly as previously described (Kleinberger et al., 2014 (link)). Microglia isolated from 3 or 6 month old APPPS1, APP-KI and WT mice were plated onto 24 well plate at a density of 2 × 10⁵ cells per well and cultured for 24 hr in a humidified 5% CO₂ incubator at 36.5°C in DMEM/F12 media (Invitrogen) supplemented with 10% heat inactivated FCS (Sigma), 1% Penicillin-Streptomycin (Invitrogen) and 10 ng/mL GM-CSF (R&DSystems). After 24 hr, plating media were replaced with fresh media. After 5 days in culture, microglia were incubated with 50 µL of E. coli particle suspension (pHrodo Green E. coli BioParticles, P35366, Invitrogen) for 60 min. Cytochalasin D (CytoD, 10 µM, from 10 mM stock in DMSO) was used as phagocytosis inhibitor and added 30 min prior to addition of bacterial particles. Bacteria excess was washed four times with PBS (on ice) and microglia attached to the plate were incubated with CD11b-APC-Cy7 antibody (1:200, clone M1/70, 557657, BD) in FACS buffer (PBS supplemented with 2 mM EDTA and 1% FBS) for 30 min at 4°C. Microglia were then washed twice with PBS, scraped off in FACS buffer and analyzed by flow cytometry. Information including mice sex and biological and technical replicates is outlined in Supplementary file 7.

For the microglial isolation quality control, around 12000 cells from a CD11b-enriched and CD11b-depleted fractions were stained in suspension with CD11b-APC-Cy7 antibody (1:200, clone M1/70, 557657, BD) in FACS buffer for 30 min at 4°C. After several washes with PBS, microglia were resuspended in FACS buffer for analysis. Propidium Iodide (PI) staining was done 10 min prior to FACS analysis. Flow cytometric data was acquired on a BD FACSverse flow cytometer by gating according to single stained and unstained samples and analyzed using FlowJo software (Treestar). Mean fluorescent intensity (MFI) is represented as the geometric mean of the according fluorochrome.