Pe conjugated anti mouse ly6c

Cells were incubated for 10 min at 4°C with anti-mouse CD16/32 (1:200; Biolegend, San Diego, CA) to block nonspecific binding, and then with FITC-conjugated anti-mouse CD11b (1:200; Biolegend), PE-conjugated anti-mouse Ly6C (1:200; Biolegend), PE/Cy7-conjugated anti-mouse F4/80 (1:200; Biolegend), AF 700-conjugated anti-mouse CD11c (1:200, Biolegend), and AF 647-conjugated anti-mouse Ly6G (1:200; Biolegend) antibodies for 30 min at 4°C, followed by eFluor 780-conjugated fixable viability dye (1:1000; eBioscience, San Diego, CA). Cells were analyzed on a Gallios Flow Cytometer (Beckman Coulter, Brea, CA), and data analyzed using Beckman Coulter Kaluza version 1.2 software. The gating strategy and cell populations characterized are shown in Fig. 1.

Lung cells were resuspended in 100 μl of staining buffer (PBS containing 2% fetal calf serum and 0.02% sodium azide) and incubated with anti-mouse CD16/CD32 (1:100; Biolegend, San Diego, CA) for 10 min at 4°C to block nonspecific binding. The cells were then incubated with FITC-conjugated anti-mouse CD11b (1:100, Biolegend), PE-conjugated anti-mouse Ly6C (1:100, Biolegend), BV-421 conjugated anti-mouse CD11c (1:100, Biolegend), AF 700-conjugated anti-mouse CD45 (1:100, Biolegend) and AF 647-conjugated anti-mouse Ly6G (1:100, Biolegend) antibodies for 30 min at 4°C followed by 30 min incubation with eFluor 780-conjugated fixable viability dye (1:100; eBioscience, San Diego, CA). Cells were then washed with PBS, fixed in 3% paraformaldehyde, and analyzed using a Beckman Coulter Gallios flow cytometer (Brea, CA). Data were analyzed using Beckman Coulter Kaluza software (version 1.2) as previously described (Sunil et al., 2015 (link)).

Cells were resuspended in 100 μl of staining buffer (PBS, 2% FCS and 0.02% sodium azide) and incubated for 10 min at 4°C with anti-mouse CD16/32 (1:100; Biolegend, San Diego, CA) to block nonspecific binding. This was followed by FITC-conjugated anti-mouse CD11b (1:200; Biolegend), PE-conjugated anti-mouse Ly6C (1:200; Biolegend), PE/Cy7-conjugated anti-mouse F4/80 (1:200; Biolegend), AF 700-conjugated anti-mouse CD11c (1:200, Biolegend), and AF 647-conjugated anti-mouse Ly6G (1:200; Biolegend) antibodies for 30 min, and then with eFluor 780-conjugated fixable viability dye (1:1000; eBioscience, San Diego, CA) for an additional 30 min at 4°C. Cells were washed twice with staining buffer, fixed with 2% para formaldehyde, and analyzed on a Beckman Coulter Gallios flow cytometer. Data were analyzed using Beckman Coulter Kaluza version 1.2 software. Lung cell populations were characterized as previously described (Sunil et al., 2015 (link)). Representative examples of our gating strategy and number of events are shown in Supplementary Figure 1. In order to directly compare the two treatment groups, the same gates were used for cell population analysis in PBS and NM treated mice. Of note, subpopulations of lung cells from NM treated mice were more heterogeneous than cells from control mice.