Rpmi 1640 medium

Manufactured by Yuanye Bio-Technology

Sourced in China

RPMI-1640 medium is a commonly used cell culture medium. It is a balanced salt solution that provides nutrients and support for the growth and maintenance of various cell types in vitro.

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RPMI-1640

5 protocols using rpmi 1640 medium

Cell Viability Assay Protocol

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KYSE-150, KYSE-270, TE-1, KYSE-510, and EC109 cell lines were purchased from Shanghai Yuanye Biotechnology Co., LTD (Shanghai, China). EC109&shFTO and EC109&shControl cell lines were supported by Servicebio (Wuhan, China). All these cells were maintained in RPMI-1640 medium (Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China) with 10% foetal bovine serum (Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China) and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were cultured with compounds at different concentrations for 72 h. Next, 5 mg/mL MTT (Servicebio, Wuhan, China) was added and incubated for 4 h. DMSO was added into the system and shocked for 10 min. The absorbance at 490 nm was measured by using a multifunction microplate reader (Thermo Fisher Scientific, Waltham, MA).

Qin B., Bai Q., Yan D., Yin F., Zhu Z., Xia C., Yang Y, & Zhao Y. (2022). Discovery of novel mRNA demethylase FTO inhibitors against esophageal cancer. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 1995-2003.

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Protosappanin B Anti-Cancer Protocol

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Protosappanin B was purchased from Shanghai Yuanye Bio-Technology Co. Ltd. (B21622-20 mg, Shanghai, China). Reagents, signaling pathway agonists and antibodies were purchased from the following companies: RPMI-1640 medium, DMEM medium (Shanghai Yuanye Bio-technology, Shanghai, China); Streptavidin Peroxidase immunohistochemistry kit (GS4961, GS4962), cell counting kit-8 (QN1293), Annexin V-FITC/PI double staining apoptosis detection kit (SNM530), and ECL western blotting detection kit (QN1155) (Beijing BioRab Technology Co. Ltd., Beijing, China); BCA protein assay kit (C05-02001), western blocking buffer (5% BSA, C05-06002) (Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China); IGF-1 (K002504P), AZD2858 (A126819), and TPA (bs-1545R) (Shanghai Hengfei Biotechnology Co. Ltd. Shanghai, China); Anti-GOLPH3 (ab98023), Anti-p-AKT (ab8805), Anti-AKT (ab38449), and anti-PCNA (proliferating cell nuclear antigen) (ab18197) (Abcam, Cambridge, UK); Anti-p-p70S6K (ribosomal protein S6 kinase, 70 kDa) (sc-8416), Anti-p70S6K (sc-8418), Anti-β-catenin (sc-7963), Anti-p-ERK1/2(extracellular signal-regulated kinase 1 and 2) (sc-81492), Anti-ERK1/2 (sc-514302), Anti-GAPDH (sc-365062), and secondary antibodies (sc-516102, sc-2357) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Zheng X.C., Shi Z.S., Qiu C.Z., Hong Z.S., Wang C.X., Zhuang H.B., Chen Z.C, & Pan J.P. (2020). Protosappanin B Exerts Anti-tumor Effects on Colon Cancer Cells via Inhibiting GOLPH3 Expression. Integrative Cancer Therapies, 19, 1534735420972477.

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Serum Shock-Induced Cell Synchronization

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Serum shock-induced synchronisation was performed based on a previously described method (21 (link), 22 (link)). In brief, 16HBE cells were seeded in medium plates with 1×10⁶ cells/plate and cultured in serum-free RPMI 1640 medium (Biological Industries, Haemek, Israel) for 24 h prior to the experiments. On the day of the experiments, the culture media were replaced with RPMI 1640 medium containing 50% horse serum (Yuanye, Shanghai, China) for 2 h. The media were then replaced with serum-free RPMI 1640. The cells were harvested and assayed at different time points (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, and 44 h) following serum shock and labelled as ZT0–ZT44, respectively.

Tang L., Liu L., Sun X., Hu P., Zhang H., Wang B., Zhang X., Jiang J., Zhao X, & Shi X. (2022). BMAL1/FOXA2-induced rhythmic fluctuations in IL-6 contribute to nocturnal asthma attacks. Frontiers in Immunology, 13, 947067.

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Enzymatic Characterization for Glucose Inhibition

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Alcalase 2.4L (2.4 AU-A/g) from Bacillus licheniformis was procured from Nanjing Chengna Chemical Company Limited (Nanjing, PR China), bromelain (300 U/mg) from pineapple, Flavourzyme (20 U/mg) from Aspergillus oryzae, trypsin (250 USP U/mg) from bovine pancreas, α-amylase (50 U/mg) from Bacilus subtilis, α-glucosidase (50 U/mg) from Saccharomyces cerevisiae, p-nitrophenyl-α-d-glucopyranoside (pNPG, ≥99%), 2,6-di-tert-butyl-4-methylphenol (BHT, ≥99%), acarbose (≥98%), RPMI 1640 medium and 10% fetal bovine serum (FBS) were procured from Shanghai Yuanye Biotechnology Company Limited (Shanghai, PR China). HT-29 cell was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, PR China). All other used reagents were of high purity and analytical grade.

Islam M.S., Hongxin W., Admassu H., Mahdi A.A., Chaoyang M, & Wei F.A. (2021). In vitro Antioxidant, Cytotoxic and Antidiabetic Activities of Protein Hydrolysates Prepared from Chinese Pond Turtle (Chinemys reevesii). Food Technology and Biotechnology, 59(3), 360-375.

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Prostate Cancer Cell Line Cultivation

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Prostate cancer cell lines (PC3, DU145 and C42B) were obtained from Shanghai Yuanye Bio-Technology Co., LTD (Shanghai, China). These prostate cancer cells were cultured in RPMI1640 medium (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China) with 10% fetal bovine serum (Shanghai Yuanye Bio-Technology Co., LTD, Shanghai, China). MDV3100-resistant prostate cancer (PC3/MDVR, DU145/MDVR and C42B/MDVR), DU145&shLSD1 and DU145&shControl cell lines were established according to reported references [8, 26, 27] . All cancer cell lines were maintained at 37 o C under 5% CO 2 .

Yan G., Zhang H., Li Y., Miao G., Liu X, & Lv Q. (2023). Viscosalactone B, a natural LSD1 inhibitor, inhibits proliferation in vitro and in vivo against prostate cancer cells. Investigational new drugs, 41(1).

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