CD204 antibody | 2F8

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding.	Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO).	Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Upstream tips
Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding.
Protocol tips
Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO).
Downstream tips
Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Publication protocol

Single-cell suspensions were incubated with a Fc receptor block (1 μg/1 × 106 cells; BD Bioscience, San Diego, CA) to reduce nonspecific anti-body binding. The panel of antibodies used in these experiments included CD45-APC-Cy7 (clone 30-F11), CD11b-V450 or PE-Cy7 (clone M1/70), CD86-PE-Cy7 (clone GL1), Gr1-PerCP-Cy5.5 (clone RB6-8C5), and streptavidin-V500 (all from BD Bioscience); CD68-FITC (clone FA-11) and CD204–Alexa Fluor 648 (clone 2F8) from AbD Serotec (Raleigh, NC); CD14-PE-Cy7 (clone Sa14-2), CD103-APC (clone 2E7), and CD206-APC (clone C068C2) from Biolegend (San Diego, CA); F4/80-PE (clone BM8) from Invitrogen (Carlsbad, CA); and CD11c-PE-Cy5 (clone N418) and MHCII–biotin (clone NIMR-4) from e-Bioscience (San Diego, CA). Dead cells were excluded using a Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO). Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Bioscience), and data were analyzed with FlowJo software (TreeStar, Ashland, OR). For morphological characterization, cells were sorted by FACS, loaded onto slides using a Cytospin 2 (Shandon Southern Products, Runcorn, Cheshire, United Kingdom), and stained using a modified Wright formula (American Scientific Products, McGaw Park, IL).

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