Fc receptor block

Retro-orbital blood was collected 2 hours after SAL or LPS injection.
Whole blood was washed and lysed with red cell lysis buffer (Biolegend, San
Diego, CA) for 5 minutes. Cells were stained for 30 minutes at 4°C with
Brilliant Violet 421-labeled anti-Ly-6G (1 μg/ml) and Alexa Fluor
647-labeled anti-CD11b antibodies (0.8 μg/ml) (both Biolegend) with 4
μL Fc receptor block per sample (BD Biosciences, San Jose, CA). Cells
were later fixed in 2% paraformaldehyde for 30 minutes^{34 (link)}, and fluorescence was measured using a
BD LSR II Flow Cytometer (BD Biosciences). Data were collected for 10,000
events. Forward vs. side scatter plots (FSC-area
vs. SSC-area) were used to identify white blood cells and
exclude cell debris. Gates were then manually drawn on plots of FSC-height
vs. FSC-area plots to gate for single cells.
Two-dimensional plots of Ly-6G vs. CD11b were used to identity
Ly-6G^hi/CD11b^hi PMNs, and histograms were constructed
for each PMN population. Geometric mean fluorescence intensity (MFI) analyses
were performed using FlowJo analysis software (TreeStar, Ashland, OR).

All stains were performed in 96 well u-bottom plates in a volume of 100 μl. 1×10⁵–1×10⁶ cells were plated and blocked with Fc receptor block (BD, Biosciences, San Jose, CA) for 15 min at 4°C and subsequently incubated with antibodies for 1 h at 4°C. Cells were then washed and fixed in 5% formalin for 15 min at room temperature. Flow cytometry was acquired using a FACSCanto RUO system (BD Biosciences). Data analysis was performed using FlowJo (Treestar, Ashland OR). Antibodies included: Siglec F PE (BD), Ly6G APC, Ly6G FITC, CD11b PE-Cy7, CD11c PB, CD71 PerCP Cy5.5, CD206 BV605, ICAM1 FITC, CD45 APC-Cy7, MHCII BV510, CD103 APC (Biolegend).

Three million cells per sample were added to BD Falcon 5 mL polypropylene round-bottom tubes. Cells were washed with 2 mL of 1X PBS and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and cells were washed in 2 mL of FACS buffer (1X PBS + 3% FBS). Supernatant was removed and 3 μL of Fc Receptor Block (BD) was diluted in 50 μL of FACS buffer per sample and incubated for 15 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and antibodies were diluted in 50 μL of FACS buffer and incubated for 30 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. If biotinylated antibody was used, streptavidin was diluted in 100 μL FACS buffer per sample and incubated for 30 minutes at 4°C. Viability staining was performed by adding Tonbo Ghost Dye UV450 (1:500) or DAPI (1:1000). Cells were analyzed on the 5-Laser BD LSR II or sorted on the 5-Laser FACS Aria III in the VUMC Flow Cytometry Shared Resources.