Hyaluronidase haase

Macrophages were seeded on coverslips and treated with 15 U/ml or 30 U/ml hyaluronidase (HAase; Sigma-Aldrich) 2 h before the experiment. For staining, cells were fixed with 4% PFA and permeabilized in 0.2% Triton X-100/PBS for 10 min and then blocked in 1% BSA, 0.1% Triton X-100 in PBS for at least 2 h. Macrophages were incubated with 3 μg/ml biotinylated hyaluronan-binding protein (Amsbio) overnight at 4°C, then washed three times with PBS. Samples were incubated with Alexa Fluor 488 Streptavidin (diluted in PBS; Biolegend) overnight at 4°C, protected from light, then rinsed three times for at least 10 min each with PBS. Coverslip were then mounted on slides using Vectashield+DAPI (Vector Laboratories).

Chemicals for the preparation of the growth medium, 2,2-diphenyl-1-picrylhydrazyl(DPPH), pyrogallol, Tris-HCl, EDTA-Na₂, 2,4,6-tripyridyl-S-triazine (TPTZ), FeCl₃·6H₂O, ascorbic acid, butylhydroxytoluene (BHT), Trolox, hyaluronic acid (HA) sodium salt from Streptococcus equi, hyaluronidase (HAase) from bovine tests, and 4-(dimethylamino)benzaldehyde (DMAB) were purchased from Sigma-Aldrich Co. (Milano, Italy). High-performance liquid chromatography (HPLC)-grade methanol and ethyl acetate were obtained from Fisher Chemical (Fisher Scientific Italia, Rodano, Milan, Italy). HPLC-grade water (18.2 mΩ) was prepared using a Millipore Milli-Q purification system (Millipore Corp., Bedford, MA, USA).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342 and DAPI were obtained from Life Technologies (Shanghai, China). Collagenase IV, DNase, hyaluronidase (HAase) were purchased from Sigma-Aldrich (Shanghai, China). Tumor penetrable Fmoc-KRGDK (FK) peptide and a tumor non-penetrable analog Fmoc-KRGDC (FC) peptide were synthesized by solid phase chemistry. Indocyanine Green (ICG) was purchased from Frontier Scientific Inc. (Logan, USA). JQ1 was ordered from Selleck Chemicals (USA). Coumarin 6 (C6) was obtained from J&K Chemicals (Shanghai, China). Anti-PD-L1 antibody was ordered from Abcam (Shanghai, China). ELISA kits for TNF-α, IFN-γ and IL-6 assay were all purchased from Neobioscience Co., Ltd. (Shenzhen, China). Phorbol-12-myristate-13-acetate (PMA) and Ionomycin were purchased from Dakewe Biotech Co., Ltd. (Shenzhen, China). All other regents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification.