Anti ly6g pe

The ARDS model was established as described previously (40 (link)). Briefly, the mice were anesthetized with sevoflurane (Hengrui, Lianyungang, Jiangsu, China). After exposing the trachea, a trimmed sterile 31-gauge needle was inserted into the tracheal lumen. LPS (Sigma, St Louis, MO, USA) diluted in endotoxin-free saline was intratracheally (IT) injected at a dose of 10 mg/kg in 100 μl saline. To treat ARDS mice, anti-PD-L1 antibody (eBiosciences, San Diego, CA, USA) was intraperitoneally administered at a dose of 50 μg/mouse immediately after the injection of LPS. Hematoxylin-eosin staining was conducted to quantify lung injury and the result was semi-quantified by two independent pathologists according to the criteria reported previously (41 (link), 42 (link)). The cells in the BALF (BALF was obtained by intratracheal injection with 1 ml cold PBS) were collected and stained with anti-Ly6G-PE and anti-CD11b-APC (eBiosciences, San Diego, CA, USA) to detect neutrophils by flow cytometry. BALF levels of TNF-α, IL-1β and IL-6 were detected by enzyme linked immunosorbent assay (ELISA, R&D, Minneapolis, MN, USA). The wet-to-dry weight (W/D) ratio was calculated to assess the edema. The protein concentration in the BALF was assessed with a BCA detection kit (Thermo Scientific, Rockford, IL, USA).

The following antibodies were used in this study: anti-CD11b-FITC (Biolegend, 101206, dilution ratio: 1:100 for flow cytometry and immunofluorescence); anti-Ly-6G-PE (eBioscience, 12-9668-82, dilution ratio: 1:100 for flow cytometry); anti-Ly-6C-PE (Cell eBioscience, 12-5932-82, dilution ratio: 1:100 for flow cytometry); anti-p-Smad1/5 (Cell Signaling Technology, 9516, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-Smad1/5 (Cell Signaling Technology, 6,944, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-p-Stat3 (Cell Signaling Technology, 9,145, dilution ratio: 1:1000 for Western blot); anti-Stat3 (Cell Signaling Technology, 9,139, dilution ratio: 1:1000 for Western blot); anti-p-Erk (Cell Signaling Technology, 4,370, dilution ratio: 1:1000 for Western blot); anti-Erk (Cell Signaling Technology, 4,695, dilution ratio: 1:1000 for Western blot); anti-mouse IL6 (Biolegend, 504501, dilution ratio: 1:500 for neutralization); anti-S100A9 (Abcam, GB111149, dilution ratio: 1:2000 for IHC); anti-Ki67 (Servicebio, GB13030-2, dilution ratio: 1:500 for IHC), and anti-β-actin (Sigma, A1978, dilution ratio: 1:1000 for Western blot). Rat tail collagen I (Corning, 356236) and RhBMP2 (R&D, 355-BM-100) were also used. Tris–HCl, NaCl, and other chemicals were from Sigma.

Cells isolated from tumor tissues were blocked with purified CD16/32 antibody for 10 min and then stained for MDSCs using anti-CD11b APC, anti-CD45 PE C-Y7, anti-Ly6G PE, and anti-Ly6C FITC antibodies (eBioscience, San Diego, CA) for 30 min on ice. For Tregs staining, the cells were first stained for extracellular Treg markers (CD4 and CD25) with anti-mouse CD4-FITC,- and anti-mouse CD25-PE for 30 min. The cells were then fixed using the FOXP3/Transcription factor staining buffer set (eBioscience cat.# 00–5523) according to the manufacturer’s protocol. The cells were subsequently washed and stained with DAPI for 10 min before analysis by flow cytometry and Flowjo software V10.

The cells in the BALF were stained with the anti-Ly6G-PE and anti-CD11b-APC antibodies (eBiosciences, San Diego, CA, USA) for cell calculation by flow cytometry operated on FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA).

Cell suspensions derived from organs or tumor homogenates from IL-15^−/− or IL-15^+/+ mice were blocked with mouse BD Fc Block (Rat anti-mouse CD16/32; BD Pharmingen) for 10 minutes and stained with anti-CD16/32-FITC, anti-CD4-APC, anti-CD8-PE, and anti-Ly6G-PE antibodies (eBioscience) for 30 minutes at 4°C, washed, filtered and analyzed on a BD FACSCalibur (Becton Dickinson).

Splenocytes were obtained from uninfected and infected animals and after RBC lysis, cells were washed in PBS, incubated with Live/Dead Fixable cell stain (ThermoFisher) for cell death exclusion, and then, with the following monoclonal antibodies: anti-CD45-APC-Cy7, anti-CD11-b-PE-Cy7, anti-Ly6C-eFluor450 and anti-Ly6G-PE (eBioscience). Samples were washed and fixed with paraformaldehyde 4% (Sigma-Aldrich) and data were acquired on a FACS LSR Fortessa flow cytometer (Becton Dickinson). Data were analyzed with FlowJo V10.0.2 (Tree Star). A forward scatter area (FSC-A) versus forward scatter height (FSC-H) gate was used to remove doublets. Viable leukocytes were selected using a Live/Dead versus CD45 gate. Expression of Ly6C and Ly6G was used to define neutrophils (Ly6C^intLy6G^high) and monocytes (Ly6C^highLy6G^int) within CD11b⁺ cells. GraphPad PrismV8.0 (GraphPad-Software) was used for graphic representation.