Fc blocking antibody 2.4 g2

In situ lung neutrophil infection was analyzed in mice on day 1 or 23 after infection with 5x10⁶ CFU of the green fluorescent BCG strain Myc409 [7 (link)]. Freshly collected lungs were incubated overnight at 4°C in 4% paraformaldehyde, 10% sucrose (w/vol in PBS) and then immersed in 30% sucrose (w/vol in PBS) for 4 h. Lung tissue was snap frozen in OCT compound (CellPath, Powys, UK). 8 μm-thick lung sections collected on Superfrost Plus slides (Thermo Fischer Scientific) were air-dried and stored at -80°C. Cryosections were blocked for 30 min with Fc-blocking antibody 2.4 G2 (BD Biosciences), washed in PBS with 2% FCS and incubated overnight at 4°C with anti-Ly-6G (clone1A8), (BD Biosciences) followed by incubation with Alexa 594 conjugated anti-rat (Invitrogen) and anti CD3ε (145-2C11, APC conjugated, Miltenyi Biotec) for the localization of T cells and neutrophils. Slides were washed in PBS with 2% and mounted with Fluoromount-G (eBioscience) including DAPI. Images were captured with a confocal Leica TCS SP8 microscope.

Splenocytes were cultured in U-bottom 96-well plates (VWR) at 1 × 10⁶ cells/200 μl of supplemented RPMI medium and incubated as described for antigen processing assays. Thereafter, cells were washed and incubated for 5 min at 4 °C with Fc blocking antibody (2.4G2) (BD Pharmingen) before stained for 20 min in the dark at 4 °C with saturating concentrations of surface-targeted antibodies and viability markers. Cells were fixed with Cytofix/Cytoperm kit (BD Biosciences), measured by FACS Fortessa (BD Biosciences) and analysed by FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded using Zombie Yellow (Biolegend). Antibodies used as follows: CD3-APC (145-2c11, Biolegend), F4/80-AF700 (cat. MCA497A700, BioRad), LY6G/6C-APC-Cy7 (RB6-8C5, BD), NK1.1-BV421 (PK136, Biolegend), MHCII-BV711 (M5/114, BD), CD11c-BV785 (N418, Biolegend), CD11b-PE-Cy7 (M1/70, BD), CD45R/B220-V500 (RA3-6B2, BD), CD103-PE (2E7, Invitrogen), CD8-Percp-Cy5.5 (53–6.7, Biolegend) and CD317-BV650 (927, Biolegend).