Pe cy7 conjugated anti ly6g

Isolated peritoneal cells were washed with PBS and stained with the following fluorescent probes obtained from BioLegend: BV570-conjugated anti-CD11b (0.66 µg/ml; 101233), APC/Fire750-conjugated anti-Ly6c (0.67 µg/ml; 128046), PE/Cy7-conjugated anti-Ly6g (0.5 µg/ml; 127617), AF488-conjugated anti-F4/80 (1 µg/ml; 123120), APC-conjugated anti-B220 (0.5 µg/ml; 103212), PerCP/Cy5.5-conjugated anti-CD3 (0.67 µg/ml; 100217), and AF700-conjugated anti-CD11c (1.67 µg/ml; 117319). Flow cytometry analysis of peritoneal cells was performed on a Gallios flow cytometer (Beckman Coulter). The data were analyzed using Kaluza 2.1 software (Beckman Coulter), and electronic compensation was applied to eliminate bleed-through fluorescence. Cell subsets were then determined via receptor expression and FSc/SSc exclusion as follows: B cells (B220⁺), T cells (CD3⁺), granulocytes (CD11b⁺, Ly6c⁺, Ly6g⁺), small macrophages (CD11b⁺, Ly6c⁺, Ly6g⁻, F4/80^int, smaller FSc/SSc), and large macrophages (CD11b⁺, Ly6c⁺, Ly6g⁻, F4/80^high, larger FSc/SSc). Cells that were negative for all markers were described as “unidentified cells”.

Cells collected from peritoneal lavage fluids was washed twice by cold PBS, and then stained with FITC-conjugated anti-CD11b (Biolegend, San Diego, CA, USA) and PE/Cy7-conjugated anti-Ly6G (Biolegend, San Diego, CA, USA) antibodies diluted in PBS with 2% FBS for 30 min at 4°C. The cells were washed two times with PBS/2% FBS. Finally, CD11b⁺ PECs and CD11b⁺ Ly6G⁺ neutrophils were analyzed by flow cytometer (CytoFLEX, Beckman Coulter, USA).

Lung digests were obtained by incubation with 1 mg/mL collagenase A, and 100 ng/mL DNase (Sigma) for 1 hour. 0.5‐1 × 10(6) cells from lung digests and BAL were stained with cell surface antibodies including PerCP‐cy5.5‐conjugated anti‐F4/80, PE‐Cy7‐conjugated anti‐Ly6G, PE‐conjugated anti‐CD45, BV421‐conjugated anti‐Siglec F and APC‐conjugated anti‐CD206 (BioLegend). Analysis was performed on FACScan cytometer (Becton Dickinson). All data were analysed on FlowJo software (Tree Star).