Cd11b pe m1 70

C57BL/6J mice aged 6–8 weeks were intraperitoneally injected with 700 mg of alum and evaluated 12 h later. The peritoneal cavity was washed with 5 ml of PBS. Peritoneal exudate cells (PECs) were analyzed by flow cytometry. The recruitment of neutrophils was visualized with CD11b-PE (M1/70; BD Biosciences) and Ly-6G-APC (1A8; eBioscience). PI (Sigma) was used to exclude dead cells. Neutrophil (CD11b⁺Ly6G⁺) recruitment was analyzed on a CytoFLEX (Beckman Coulter) (45 (link)).

4T1 tumours were excised from CSG and TNFα‐CSG‐treated mice, minced and incubated in DMEM high‐glucose medium containing 0.1 mg/ml DNase I (Sigma) and 0.5 mg/ml collagenase IV (Sigma) for 1 h at 37°C under gentle rotation. The cell suspension was passed through a 70‐μm membrane and subsequently washed with FACS buffer [1% BSA (w/v, Sigma) in PBS]. For analysis of immune cells, viable cells (Zombie Aqua™ selection; BioLegend) were stained with the following antibodies: CD45‐APC‐efluor 780 (30‐F11; eBioscience, 1:200), CD3‐BUV395 (145‐2C11; BD, 1:200), CD4‐BV510 (Gk1.5; BD, 1:100), CD8‐PE‐CF594 (53‐6.7; BD, 1:300), CD11b‐PE (M1/70; BD, 1:300), F4/80‐AF647 (BM8; BioLegend, 1:300) and Ly6G‐FITC (1A8; BD, 1:100). For analysis of cytotoxic and regulatory T cells, viable cells were stained with the following antibodies: CD3‐APC‐eFluor 780 (145‐2C11; eBioscience, 1:200), CD4‐FITC (Gk1.5; eBioscience, 1:200), CD8‐PerCp‐eFluor 710 (53‐6.7; eBioscience, 1:200), CD25PE‐efluor 610 (PC61.5; eBioscience, 1:100), FoxP3‐AF647 (150; BioLegend, 1:100) and granzyme B (GB11; BD, 1:100). For intracellular staining (granzyme B and FoxP3), cells were permeabilised with True‐Nuclear™ Transcription Factor Buffer (Biolegend). Cells were sorted on a BD SORP Fortessa and analysed on BD FACSDiva software version 8.0.1 (BD Biosciences, USA).

Neutrophil granulocytes were depleted by i.p. injection of 500 μg anti-Ly6G-antibody, clone 1A8 (BioXCell, #BE0075-1) and corresponding isotype control (InVivoMab Rat IgG2a, clone 2A3, BioXCell, #BE0089) 16 h prior to osmotic mini pump implantation.
For flow cytometric analysis of circulating neutrophils EDTA anticoagulated blood was collected 48 h after implantation of osmotic mini pumps and stained followed by lysis of red blood cells. Cells were fixed in 1% neutral buffered paraformaldehyde for 20 min at room temperature, according to a protocol by [6 (link)]. To prevent unspecific binding, cells were incubated with anti-CD16/32 antibody (clone 93; Biolegend, San Diego, CA, USA) before antibody staining.
CD11b-PE (M1/70, BD Biosciences, San Jose, CA, USA) and anti-Ly-6C-AlexaFluor^® 488 (HK1.4) were used to detect neutrophils and monocytes [21 (link)]. Respective isotype control IgGs were used to set all flow cytometric gates. Measurement and data analysis were performed using a Gallios™ Flow Cytometer, and Kaluza^® Flow Analysis Software (both Beckman Coulter Inc., Krefeld, Germany).