Rat anti mouse f4 80 apc

Single cell suspensions of macrophages were taken immediately after harvest or following culture by removal of the adherent macrophages by a 2 minute incubation at 37°C in 0.05% trypsin/0.53 mM EDTA (Corning) followed by gentle mechanical lifting from the plate with a sterile cell scraper. Cells were then washed with PBS and nonspecific binding was blocked by a 10 minute incubation at 4°C with anti-mouse CD16/32 antibody (TruStain FcX, BioLegend). Cells were again washed with PBS and then incubated for 20 minutes at 4°C with the fluorochrome conjugated antibody of choice for that experiment: rat anti-mouse F4/80 APC (BD Biosciences clone T45-2342), APC rat IgG2a,k isotype control antibody (BD Biosciences), hamster anti-mouse CD11c PE (BD Pharmingen clone HL3), PE Armenian hamster IgG1,l2 isotype control antibody (BD Pharmingen), rat anti-mouse I-A/I-E PE (BD Pharmingen clone M5/114.15.2), or PE rat IgG2b,k isotype control antibody (BD Pharmingen). Cells were then washed once more in PBS and resuspended in 1% paraformaldehyde for analysis by the NIH Cancer and Inflammation Program Flow Cytometry Core. Data were analyzed with FlowJo software.

For extracellular staining, 10⁶ peritoneum cells were stained with the following antibodies: hamster anti-mouse CD3ϵ FITC (isotype hamster IgG1, κ), rat anti-mouse CD49b PE (isotype rat IgM, κ, BD Biosciences), rat anti-mouse CD19 Percp-Cy5.5 (isotype rat IgG2a, κ, BD Biosciences), and rat anti-mouse CD138 APC (isotype rat IgG2a, κ, BD Biosciences). In parallel, 10⁶ peritoneum cells were used to determine the monocyte phenotype, using rat anti-mouse CD11b PE (isotype rat IgG2b, κ, BD Biosciences) and rat anti-mouse F4/80 APC (isotype rat IgG2a, κ) antibodies.
Data were read in a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with the software CellQuest (Becton Dickinson).