The normal renal cell line HK-2, human renal cancer cell line 786-O and Caki-1 were all from the cell bank of the typical Culture Committee of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 medium, McCoy’s 5A medium, trypsin, streptomycin, and penicillin were purchased from Wuhan Boster Biological Technology, LTD. (Wuhan, China). Fetal bovine serum (FBS) was purchased from GIBCO (Grand Island, New York, United States). The medium of HK-2 and 786-O cell lines contained 90% RPMI 1640, 10% FBS, and 1% antibiotics (100 g/ml streptomycin and 100 U/ml penicillin). Caki-1 was cultured in 90% McCoy’s 5A supplemented with 10% FBS and 1% streptomycin and penicillin. All cell lines were cultured in 5% CO2 at 37°C. The culture medium was renewed every 2–3 days. Experiments were then performed on passaged three to five cells.
Rpmi 1640 medium
Manufactured by Wuhan Boster Biological Technology
Sourced in China
About the product
RPMI-1640 medium is a widely used cell culture medium, originally developed for the growth of human leukemic cells. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids to support the growth and maintenance of a variety of cell types, including immune cells, cancer cells, and other mammalian cell lines.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Wuhan Boster Biological Technology (1 mentions)
Df13236, by Affinity Biosciences (1 mentions)
A00227 1, by Wuhan Boster Biological Technology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Mtt agent, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Penicillin, by Tianjin Haoyang Biological Manufacture (1 mentions)
Fetal bovine serum (fbs), by Tianjin Haoyang Biological Manufacture (1 mentions)
Streptomycin, by Tianjin Haoyang Biological Manufacture (1 mentions)
Cucurbitacin a, by Merck Group (1 mentions)
Hoechst, by Wuhan Boster Biological Technology (1 mentions)
33258 dmem, by Wuhan Boster Biological Technology (1 mentions)
Trypsin, by Wuhan Boster Biological Technology (1 mentions)
Mccoy s 5a medium, by Wuhan Boster Biological Technology (1 mentions)
Streptomycin, by Wuhan Boster Biological Technology (1 mentions)
Penicillin, by Wuhan Boster Biological Technology (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Htr 8 svneo cells, by American Type Culture Collection (1 mentions)
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Product FAQ
11 protocols using «rpmi 1640 medium»
1
Cell lines and culture conditions
2
Overexpression of P2RX2 in Prostate Cancer
Two PCa cell lines (CWR22Rv1 and C4-2b) were purchased from the Shanghai Institute of Cell Biology (Shanghai, China) and Wuhan Shanen Biotechnology (Wuhan, China). The cell lines were cultured in RPMI1640 medium (Wuhan Boster Biological Technology, China) with 10% fetal bovine serum (Gibco, Invitrogen, Shanghai, China) and incubated at 37°C in a 5% CO2 incubator at a constant temperature. C4-2b and CWR22Rv1 cells were seeded in 6-well plates for transfection. P2RX2-overexpressing plasmids and corresponding control vectors were generated by Wuhan Viral therapy Technologies (Wuhan, China), and the effective sequences are presented in Table S1 . P2RX2-overexpressing plasmids and no-load control plasmids were transfected using Lipofectamine 3000 (Invitrogen, Shanghai, China). Thereafter, protein samples were extracted from the transfected cells, subjected to SDS-PAGE, separated by electrophoresis, and transferred onto PVDF membranes. The membranes were blocked in 5% BSA and subsequently incubated with primary antibodies against P2RX2 (DF13236, Affinity) and GAPDH (A00227-1, Wuhan Boster Biological Technology) at 4°C overnight. The membranes were then washed thrice with TBST three times and incubated with secondary antibodies at room temperature for 1 h.
To validate the tumor-suppressive role of P2RX2 in PCa, cell activity was detected using a cell counting kit-8 (CCK-8) assay (C0037, Beyotime). The cells were seeded into 96-well plates, and a CCK-8 solution was added to each well after 24 h. The absorbance of each well at 450 nm was measured after 2.5 h. Next, a colony formation assay was performed. The cells were seeded in 6-well plates (2 × 104/well) and cultured for 2 weeks. The cells were fixed in formaldehyde and stained with crystal violet, and cell clones were counted. Finally, a scratch assay was performed. The cells were seeded in 24-well plates and incubated until 100% confluence was reached. After 72 h, the changes in cell migration were estimated.
To validate the tumor-suppressive role of P2RX2 in PCa, cell activity was detected using a cell counting kit-8 (CCK-8) assay (C0037, Beyotime). The cells were seeded into 96-well plates, and a CCK-8 solution was added to each well after 24 h. The absorbance of each well at 450 nm was measured after 2.5 h. Next, a colony formation assay was performed. The cells were seeded in 6-well plates (2 × 104/well) and cultured for 2 weeks. The cells were fixed in formaldehyde and stained with crystal violet, and cell clones were counted. Finally, a scratch assay was performed. The cells were seeded in 24-well plates and incubated until 100% confluence was reached. After 72 h, the changes in cell migration were estimated.
3
Quantitative Wound Healing and Invasion Assays
For wound healing assays, cells at the logarithmic growth phase were cultured to reached 100% confluence in 6-well plates. The next day, the AsPC-1 cell layer was scratched with a 10-µl micropipette tip in the center of the well (denoted as 0 h). DMEM with 1% FBS was used to avoid cell proliferation. The AsPC-1 cells were gently rinsed with PBS and incubated in 1% FBS-containing DMEM. After 24 h of incubation at 37°C, the cells were removed from the incubator, photographed using an inverted light microscope (magnification, ×200; Leica Microsystems, Inc.), and the cell migration distance was measured. The width of the wound healing site was quantified and compared with baseline values. All experiments were repeated independently in triplicate.
Cancer cell invasion was tested using Transwell assays. Cells were removed from serum-containing medium and serum-starved for 24 h using serum-free medium. Matrigel (BD Biosciences) was thawed at 4°C overnight, and 2×104 cells from each group in 200 µl serum-free medium were seeded in the upper chamber (pore size, 8.0 µm; Corning, Inc.) precoated with 90 µl Matrigel at 37°C for 8 h. Subsequently, 600 µl RPMI-1640 medium (Wuhan Boster Biological Technology Co., Ltd.) containing 10% FBS was added to the lower chamber. After 24 h of incubation at 37°C, the upper chambers were fixed with 4% polymethanol (Wuhan Boster Biological Technology Co., Ltd.) for 30 min at room temperature, and then stained with 0.1% crystal violet for 30 min at room temperature. The cells that migrated through the membrane and invaded the underside of the upper chamber were photographed using an inverted light microscope (magnification, ×200; Leica Microsystems, Inc.). Five random fields were selected to calculate the number of migrating or invading cells.
Cancer cell invasion was tested using Transwell assays. Cells were removed from serum-containing medium and serum-starved for 24 h using serum-free medium. Matrigel (BD Biosciences) was thawed at 4°C overnight, and 2×104 cells from each group in 200 µl serum-free medium were seeded in the upper chamber (pore size, 8.0 µm; Corning, Inc.) precoated with 90 µl Matrigel at 37°C for 8 h. Subsequently, 600 µl RPMI-1640 medium (Wuhan Boster Biological Technology Co., Ltd.) containing 10% FBS was added to the lower chamber. After 24 h of incubation at 37°C, the upper chambers were fixed with 4% polymethanol (Wuhan Boster Biological Technology Co., Ltd.) for 30 min at room temperature, and then stained with 0.1% crystal violet for 30 min at room temperature. The cells that migrated through the membrane and invaded the underside of the upper chamber were photographed using an inverted light microscope (magnification, ×200; Leica Microsystems, Inc.). Five random fields were selected to calculate the number of migrating or invading cells.
4
Culturing and Inhibition of HTR-8/SVneo Cells
HTR-8/SVneo cells (American Type Culture Collection) were cultured in RPMI-1640 medium (Wuhan Boster Biological Technology, Ltd.) supplemented with 10% FBS (Biological Industries), 100 mg/ml streptomycin and 100 U/ml penicillin. Cells were cultured in a humid atmosphere of 5% CO2 and 37˚C. Cells were extracted after reaching 80% confluence. Drug intervention cells were cultured in RPMI-1640 (Sigma-Aldrich; Merck KGaA) supplemented with 100 ng/ml IFN-γ, while control cells were cultured in conventional RPMI-1640 in a humid atmosphere of 5% CO2 and 37˚C for 48 h. For inhibition experiments, cells were subjected to fluorescein amidites-labeled small interfering RNA (siRNA) transfection and cultured in RPMI-1640 medium for 48 h. Cells were collected for analysis 24 h after transfection.
5
Protective Effects of SSd Against CCl4-Induced Liver Injury
Cell culture. HL-7702 cells (the human normal liver cell line from FuDan IBS Cell Center, Shanghai, China) were routinely cultured in RPMI-1640 medium (Wuhan Boster Biological Technology, Ltd., Wuhan, China) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 5% CO 2 at 37˚C. The cells were treated with CCl 4 (Beijing Chemical Reagent Company, Beijing, China), SSd (batch no. 110778-201409; China's Drug Supervision, Beijing, China), and N-acetyl-L-cysteine (NAC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). SSd, CCl 4 , and NAC were dissolved in dimethyl sulphoxide (DMSO) immediately before use (final concentration of DMSO: <0.1%). DMSO (<0.1%) alone was included as a control in all experiments and did not have any effect on the parameters measured.
MTT assay. Cell proliferation was analyzed by the MTT assay. HL-7702 cells were washed twice with phosphate-buffered saline solution (PBS) and counted. Then, 1x10 4 cells were seeded on 96-well plates and cultured for 24 h. The cells were exposed to SSd at various concentrations for 24 h. Other cells were seeded onto 96-well plates, incubated for 24 h, and preincubated with NAC (100 µmol/l) or the indicated doses of SSd for 1 h. After that, CCl 4 was added at 10 mmol/l to induce acute injury for 24 h (25, 26) . Then, 100 µl of MTT solution (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) were added to each well and incubated for 4 h. The medium was discarded and 150 µl of DMSO was added for 24 h. The absorbance of each well was measured at 570 nm with an ELx800 universal microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative cell viability was calculated as: (absorbance of drug treated group)/(absorbance of untreated group) x 100 (%).
Alanine aminotransferase (ALT) and aspartate transaminase (AST) in supernatants. HL-7702 cells were seeded onto 6-well plates at 8x10 5 cells/well and cultured in RPMI-1640 with 10% FBS for 24 h. After HL-7702 cells were pretreated with NAC or the indicated doses of SSd for 1 h, CCl 4 was added at 10 mmol/l to induce acute hepatocellular injury for 24 h (25, 26) . The supernatants were collected and stored at -20˚C. ALT and AST levels were measured using a Var10skan Flash fluorescence plate reader (Thermo Fisher Scientific, Inc.) using the ALT and AST assay kits (C009-1 and C010-1; Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).
MTT assay. Cell proliferation was analyzed by the MTT assay. HL-7702 cells were washed twice with phosphate-buffered saline solution (PBS) and counted. Then, 1x10 4 cells were seeded on 96-well plates and cultured for 24 h. The cells were exposed to SSd at various concentrations for 24 h. Other cells were seeded onto 96-well plates, incubated for 24 h, and preincubated with NAC (100 µmol/l) or the indicated doses of SSd for 1 h. After that, CCl 4 was added at 10 mmol/l to induce acute injury for 24 h (25, 26) . Then, 100 µl of MTT solution (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) were added to each well and incubated for 4 h. The medium was discarded and 150 µl of DMSO was added for 24 h. The absorbance of each well was measured at 570 nm with an ELx800 universal microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative cell viability was calculated as: (absorbance of drug treated group)/(absorbance of untreated group) x 100 (%).
Alanine aminotransferase (ALT) and aspartate transaminase (AST) in supernatants. HL-7702 cells were seeded onto 6-well plates at 8x10 5 cells/well and cultured in RPMI-1640 with 10% FBS for 24 h. After HL-7702 cells were pretreated with NAC or the indicated doses of SSd for 1 h, CCl 4 was added at 10 mmol/l to induce acute hepatocellular injury for 24 h (25, 26) . The supernatants were collected and stored at -20˚C. ALT and AST levels were measured using a Var10skan Flash fluorescence plate reader (Thermo Fisher Scientific, Inc.) using the ALT and AST assay kits (C009-1 and C010-1; Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).
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