Fitc conjugated anti f4 80 ab

Tumor-draining LNs were harvested from mice treated with E7-specific protein vaccine and/or anti-PD-L1 Ab 7 days after the last protein vaccination (on day 28 after tumor challenge) (Figure 4A). Single cell suspensions were prepared as described previously [47 (link)]. To detect the maturation status of DCs and the M1-like polarization of macrophages, the cells were stained with FITC-conjugated anti-CD11c Ab, FITC-conjugated anti-F4/80 Ab, PE-conjugated anti-CD80 Ab, and PE-Cy5-conjugated anti-CD86 Ab (BioLegend) or PE-conjugated anti-MHC class I and PE-Cy5-conjugated anti-MHC class II Abs (BioLegend). The cells were analyzed by flow cytometry, as described above.

BMDC infected with e-GFP L. major or stimulated with LmAg (30 µl LmAg/ml, equivalent to 30 parasites per BMDC) were fixed with 4% PFA and resuspended in FACS buffer containing the following antibodies (Ab): phycoerythrin-cyanine 7 (PECy7)–conjugated anti-CD11c (BD Biosciences, Heidelberg, Germany), phycoerythrin (PE)-conjugated anti-CD86 (BD Biosciences), allophycocyanin (APC)-conjugated anti-MHC class II (Miltenyi, Bergisch Gladbach, Germany). For some assays, BMDC were infected with WT L. major promastigotes instead, and fluorescein isothiocyanate (FITC)-conjugated anti-CD40 (Biolegend, San Diego, USA) and FITC-conjugated anti-CD80 (eBioscience, San Diego, USA) Ab were used. Data was obtained using the MACSQuant flow cytometer (Miltenyi) and analyzed using FlowJo (Tree Star Inc., CA, USA). The expression of F4/80 in BMM was determined using FITC-conjugated anti-F4/80 Ab (Biolegend).