Anti mouse inos apc

We labeled cells with antibodies against markers: anti‐mouse CD11b‐PE (clone: M1/70; BioLegend), anti‐mouse Gr‐1‐FITC (clone: RB6‐8C5; BioLegend), anti‐mouse Ly6G‐FITC (clone: 1A8‐Ly6g; eBioscience), anti‐mouse Ly6C‐APC (clone: HK1.4; eBioscience), anti‐mouse CD4‐FITC (clone: GK1.5; BD Biosciences), anti‐mouse CD4‐APC (clone: GK1.5; eBioscience), anti‐mouse IL‐17A‐PE (clone: TC11‐18H10; BD Biosciences), anti‐mouse Foxp3‐PE (clone: FJK‐16s; eBioscience), anti‐mouse CD124‐PE/Cy7 (IL‐4Rα, clone: I015F8; BioLegend), anti‐mouse CD119‐PE (IFN‐γR1, clone: 2E2; eBioscience), anti‐mouse iNOS‐APC (clone: CXNFT; eBioscience), anti‐mouse CCR2‐APC (clone: 475301; R&D Systems), and anti‐mouse CD197‐PE/Cy7 (CCR7, clone: 4B12; eBioscience). MDSCs and T lymphocytes were surface stained after 30 min at 4°C incubation with relevant antibodies shielded from light. CD4⁺ T cells were stained with anti‐IL‐17A after being stimulated with the Cell Stimulation Cocktail (eBioscience) for 4–6 h to measure intracellular cytokine production. Following incubation in 1× Fixation and Permeabilization Solution, CD4⁺ T was stained with Foxp3 antibody for Treg detection. Data were acquired on a BD FACSCalibur™ flow cytometer (BD Biosciences) and analyzed with FlowJo version 7.6.1 (Treestar).

AS-IV powder (C₄₁H₆₈O₁₄, molecular weight: 784.9702, purity > 98%) was purchased from Shanghai Ronghe Co. (Shanghai, China). The following antibodies were purchased from eBiosciences (CA, USA): anti-human CD206 PE, anti-human CD86 PE, anti-human CD14 PerCP/Cy5.5, anti-mouse CD45 v450, anti-mouse CD11b FITC, anti-mouse F4/80 PE, anti-mouse CD206 Alexa-647, and anti-mouse iNOS APC. Antibodies against total and phosphorylated AMPKα, arginase 1 (Arg-1), CD31, and vascular endothelial growth factor A (VEGFA) were purchased from Cell Signaling Technology (MA, USA). IL4, IL13, IFN-γ, and LPS were purchased from Peprotech (NJ, USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Liankebio (Hangzhou, China). JetPrime transfection agent was obtained from Polyplus (NY, USA).