3 laser novocyte flow cytometer

Before flow cytometry, single cell suspensions placed in ex vivo culture for 2 hours with GolgiStop 1:2,000 dilution (BD Biosciences). Gating strategy used selected live, lineage⁻ (CD3⁻CD19⁻Ter119⁻NK1.1⁻), non-neutrophil (Ly6G⁻), CD11b⁺ cells. Cells were then fixed with 2% formaldehyde and then washed/permeabilized with BD perm/wash buffer (BD Biosciences) for intra-cellular flow cytometry. After permeabilization, intra-cellular stain for anti-IL1β (BD Biosciences) was added. Cells were acquired on a 3-Laser Novocyte Flow Cytometer (Acea Biosciences, Inc.) and data was analyzed using FlowJo software version 10.0 (Treestar, Inc.). To verify gating and purity, all populations were routinely back-gated.

Single cell suspensions were collected and washed two times with cold PBS and filtered into a 96-well plate for surface staining. Cells were initially stained with pacific orange LIVE/DEAD fixable viability dye (Thermofisher) and then washed two times with cold PBS. Cells were then resuspended in Flow Buffer (PBS, FBS, NaN3, and Hepes Buffer) and Fc-Receptors were blocked with anti-CD16/32 (Biolegend) prior to surface staining. Biotinylated monoclonal antibodies used for surface staining included: Anti-CD3, Anti-CD19, Anti-Ter-119, Anti-NK1.1, Anti-CD11b, Anti-Ly6G, and Anti-Ly6C (Biolegend). Following surface staining, cells were washed twice, and biotinylated antibodies were labeled with streptavidin APC-Cy7. Next, cells were either washed and acquired for surface-only flow cytometry, or were fixed with 2% formaldehyde and then washed/permeabilized with BD perm/wash buffer (BD Biosciences) for intra-cellular flow cytometry. After permeablilization, intra-cellular stains included: anti-IL1β (BD Biosciences), anti-TNF-α (Biolegend), anti-iNOS (affymetrix). After washing, samples were then acquired on a 3-Laser Novocyte Flow Cytometer (Acea Biosciences, Inc.). Data was analyzed using FlowJo software version 10.0 (Treestar, Inc.) and data was compiled using Prism software (GraphPad, Inc.). To verify gating and purity, all populations were routinely back-gated.