Pe conjugated anti mouse f4 80 antibody

Mice were infected and treated with the test compounds as described above. After 12 h of infection, PLF was collected with 2 mL of sterile PBS, and the cells were centrifuged at 1800 rpm for 3 min. Then, the cells were blocked with 1 mL of 2% bovine serum albumin (BSA) in PBS for 30 min. An anti-mouse CD16/32 antibody (1:50) (BioLegend, CA, USA) was used to block nonspecific antibody binding in 0.5% BSA buffer for 30 min at 4 °C. Next, the cells were stained with PE-conjugated anti-mouse F4/80 antibody (1:50) (BioLegend, CA, USA) and FITC-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (1:200) (BioLegend, CA, USA) in 0.5% BSA for 30 min at 4 °C. Afterwards, the cells were washed twice with 150 μL of 2% BSA and analysed using flow cytometry.

Con A with undetectable endotoxin was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and injected into mice via the tail vein at a dose of 15 mg/kg. GL was purchased from Tokyo Chemical Industry (Tokyo, Japan) and administered to mice by intraperitoneal injection at a dose of 50 mg/kg 1 h before Con A injection. Recombinant mouse IL-25 was purchased from Sino Biological Inc (Wuhan, China). IL-25 was intraperitoneal injected at a dose of 100 μg per mouse 1 h before Con A injection in the in vivo experiment and at a concentration of 50 μg/ml in 1640 medium (Gibco) supplemented with 10% FBS (Gibco) in the in vitro experiment. An anti-IL-25 antibody was purchased from Abcam (Shanghai, China). The urea and blood creatinine testing kits were purchased from Nanjing Jiancheng (Nanjing, China). APC-conjugated anti-mouse TLR4 antibody and PE-conjugated anti-mouse F4/80 antibody were purchased from Biolegend (San Diego, CA, USA), and the anti-mouse CD206 antibody was purchased from Sevicebio (Wuhan, China). ELISA kit for IL-1β and IL-10 were purchased from Biolegend.

PDA tumor growth in KPC mice was monitored every month using small-animal ultrasound (Vevo770, VisualSonics, Toronto, Ontario, Canada). The tumor was harvested when reaching 10 mm in size, minced, and dissociated in pre-warmed digest medium [5% FBS RPMI 1640 with collagenase (1500 U/ml), and hyaluronidase (1000 U/ml); Life Technology, Carlsbad, CA, USA] and incubated at 37 °C for 1 h. Tumor cells after digestion were filtered through a cell strainer, centrifuged, and washed with cold PBS.
TAMs were sorted by the Flow Cytometer. The cell suspension was stained with a mixture of PE-conjugated anti-mouse F4/80 antibody (Biolegend), APC-conjugated anti-mouse CD3 antibody (Biolegend), PE-Cy™7 rat anti-mouse CD8a (BD Biosciences, San Jose, CA, USA), FITC-conjugated anti-CD4 antibody (Biolegend), and PI (BD Biosciences) for 30 min. CD3-F4/80⁺ live cells were selected for analysis.