Hematoxylin

Pure AST was provided by Shanghai Yuanye Biotechnology Co., Ltd. For experiments, AST was dissolved in phosphate buffered saline (PBS) and diluted with normal saline. Pentobarbital sodium, papain and cysteine were purchased from Guangzhou Chemical General Factory, and hematoxylin, water-soluble eosin Y, toluidine blue O, paraformaldehyde and EDTA were acquired from Takara Bio, Inc. Anti-TNF (cat. no. ab1793), IL-1 (cat. no. ab9722), β-actin (cat. no. ab8227), AKT (cat. no. ab8805), NF-κB (cat. no. ab28849), PI3K (cat. no. ab140307) and IL-6 (cat. no. ab6672) antibodies were all purchased from Abcam. The quantitative PCR detection and Prime Script RT kits were purchased from Bao Biological Engineering Co. Ltd., and Takara Biotechnology Co. Ltd., respectively.

Pure AST is provided by Shanghai Yuanye Biotechnology Co., Ltd. In the experiment, pure AST was dissolved with phosphate buffer (PBS) and diluted with saline. Pentobarbital sodium, papain, and cysteine were purchased from Guangzhou Chemical Reagent Factory. Hematoxylin, water-soluble eosin Y, toluidine blue O, paraformaldehyde, and Ethylene Diamine Tetraacetic Acid (EDTA) were from TAKARA, Japan. Anti- PEG2, β-actin, and other monoclonal antibodies were purchased from Abcam, UK. Finally, the immunohistochemical kit was purchased from Boster, Wuhan, China) The fluorescence quantitative PCR detection kit was purchased from Baoshi, Dalian, China, and the Prime Script RT kit were purchased from Takara, Dalian, China.

Trypsin was added to the culture flask to digest the cells. After the digestion was terminated, the cell suspension was pipetted, added to a centrifugal tube, and centrifuged for 5 min at 3,000 × g. The cells were washed once or twice with PBS and resuspended with the serum-free medium containing BSA until the cell density reached 1×10⁴/ml after the culture solution was discarded. The cells and the culture medium were inoculated into a Transwell 6-well microplate (Corning, Inc., Corning, NY, USA). A total of 1 ml of the culture medium containing FBS was added to the lower chamber. Then, 2 ml of the serum-free tumor cell suspension prepared in advance was added to the upper chamber. The cells were routinely cultured for 24 h. The culture medium in the upper chamber was discarded. The cells on the upper chamber side of the semipermeable membrane at the bottom were removed with a cotton swab. The cells were fixed for 15 min with 95% ethanol, stained for 10 min with hematoxylin (Takara Biotechnology Co., Ltd., Dalian, China), and observed and photographed under an inverted microscope using four fields of view (×200). The cells passed through the semipermeable membrane and entered the lower chamber. The cell migration ability was detected.