Cd11c apc clone s hcl 3

For imaging flow cytometry, macrophages were detached from tissue culture plates by incubating with 5 mM EDTA in 1× PBS pH 8 for 30 min and gentle scraping, washed once with 1× PBS, and fixed with 4% paraformaldehyde in 1× PBS for 30 min at room temperature. Cells were then washed with 1× PBS containing 0.1% BSA (bovine serum albumin) (PBS-BSA), resuspended in 50 μl of PBS-BSA containing 5 μl of Fc receptor blocking solution, FcX (BioLegend, San Diego, CA), and incubated at room temperature for 5 min. After incubation, 50 μl of PBS-BSA containing 2.5 μl of CD11c APC (clone S-HCL-3; BD Biosciences) antibody were added to each tube, and samples were incubated at 4°C for 30 min. After washing with PBS-BSA, cells were stained with 0.3 μg/ml Bodipy 493/503 (Life Technologies, Carlsbad, CA) in 1× PBS for 15 min. For each experimental condition, data from 5,000–10,000 CD11c+ cells were acquired at the ImageStream^XMark II imaging flow cytometer (Amnis Corporation, Seattle, WA) using 60 × magnification. Image data were analyzed by IDEAS software version 6.0 (Amnis Corporation, Seattle, WA) after applying a compensation matrix and selecting the region of interest (lipid droplets) with the Spot Mask tool. Median fluorescence intensity per cell was extracted.

For wells that did not receive Golgi plug, cells were harvested, centrifuged (1000 × g, 2 min) and surface‐stained with fluorescently labelled monoclonal antibodies to identify DC and monocyte populations (room temperature (RT), 15 min, dark; FITC Lineage Cocktail 2 [containing: CD3 (clone SK7), CD14 (clone MΦP9), CD19 (clone SJ25C1), CD20 (clone L27), CD56 (clone NCAM16.2)], CD45 PerCP (clone 2D1), CD11c APC (clone S‐HCL‐3), CD3 APC‐H7 (clone SK7), HLA‐DR V450 (clone L243)and CD14 V500 (clone M5E2); all BD Biosciences), followed by erythrocyte lysis (1 × FACS Lyse, BD Biosciences; RT, 10 min, dark). Leucocytes were then divided and stained with PE‐conjugated co‐stimulatory and adhesion molecules CD9 (clone M‐L13), CD38 (clone HIT2), CD40 (clone 5C3), CD80 (clone L307.4), CD83 (clone HB15e) or CD86 (clone FUN‐1); (RT, 30 min, dark; all BD Biosciences), washed (3% heat‐inactivated foetal calf serum (FCS)/phosphate‐buffered saline (PBS; both from Gibco by Life Technologies)) and resuspended in 1 × Stabilizing Fixative (BD Biosciences) for flow cytometric analysis.