Primary human umbilical vein vascular endothelium cells (HUVECs, ATCC® PCS-100-010™) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and maintained in endothelial cell growth medium including growth supplements (EGM, CC-3124, Lonza, Walkersville, MD, USA). 4T1 mouse breast cancer cell line and MDA-MB-231 human breast cancer cell line were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The 4T1 cells were maintained in RPIM1640 (Gibco, Carlsbad, CA, USA), and MDA-MB-231 cells were cultured in L-15 (Gibco). Both RPIM1640 and L-15 media contain 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL phytomycin. HUVECs and 4T1 cells were incubated at 37 °C with 5% CO2, and MDA-MB-231 cells were incubated at 37 °C without CO2.
Rpim1640
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
The RPIM1640 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a cell culture media formulation used to support the growth and maintenance of various cell types in vitro. The product provides a balanced nutrient solution designed to support the optimal growth and proliferation of cells in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Canto 2 flow cytometry system, by BD (2 mentions)
Ficoll hypaque 10 771, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Atcc pcs 100 010, by American Type Culture Collection (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Lipofectamine 2000 lip2000, by Thermo Fisher Scientific (1 mentions)
Spautin 1, by Selleck Chemicals (1 mentions)
Abt 263, by Selleck Chemicals (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
31 protocols using rpim1640
1
Culturing and Maintaining Breast Cancer and Endothelial Cells
2
Luciferase-Labeled Cell Line Generation
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The HEK293T and ovarian cancer cell lines were originally obtained from American Type Culture Collection (ATCC) or Japanese Collection of Research Bioresources Cell Bank (JCRB), where cell characterization was authenticated using polymorphic short tandem repeat (STR) profiling. No mycoplasma contamination was found. Luciferase-labeled stable cell lines were generated by infecting HEY or SKOV3 cells with lentiviral construct expressing firefly luciferase, followed by puromycin selection (5 μg/mL) for one week. Cells were maintained in RPIM1640 (Life Technologies) supplemented with 10% fetal bovine serum (Gibco). All cells were cultured at 37°C in a saturated humidity atmosphere containing 5% CO2.
3
Quantitative Protein Analysis of Glioma Cell Lines
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U87 and U251 cells were purchased from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Shanghai, China). Cells were cultured in RPIM 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Millipore). Small interfering RNAs (siRNAs) were purchased from RIBOBIO (Guangzhou, China). Lipofectamine™ 2000 (Lip2000) was obtained from Invitrogen (Carlsbad, CA) and transfections were performed according to the manufacturer’s instructions. The siRNA sequence was as follows: 5ʹ-CCAGAGGUCA GCUGUUCAAGA-3ʹ; Scramble siRNA sequence: 5ʹ-UUCGUAUCUGGGUGUAC CCTT-3ʹ. RIPA lysate was used to extract total protein from the cell lines and the protein concentration in the cell lysate was determined using the BCA protein quantification kit (Beyotime, China). SDS–PAGE was performed in 10% gel with the loading of an equal amount of protein per lane. Samples were electrophoretically transferred onto the PVDF membrane. The membranes were washed five times for 10 min with TBST and blocked using 5% non-fat dry skim milk in TBST before reblotting. The membranes were incubated overnight with primary antibodies, followed by incubation with relevant biotinylated secondary antibodies for 1 hour at room temperature. The membrane was scanned using an infrared Odyssey scanner by Li-cor.
4
Peptide Library for ESAT-6 Epitope Screening
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One hundred and three overlapping peptides spanning the complete amino acid sequence of CFP-10, ESAT-6, Rv1983p257-558, Rv1986, Rv1987, Rv1988 and Rv1989c, were designed and purchased (GL Biochem Ltd., Shanghai) (Table S1 ). The identity of each peptide was confirmed by mass spectrometry and the purity (>90%) was checked by high-pressure liquid chromatography. The stock concentration (20 mg/mL) of the peptides was prepared in DMSO (Sigma), and the peptides were further diluted to a working concentration in tissue culture medium RPIM-1640 (Life Technology). An ESAT-6 peptide pool containing ESAT-6 P1-P8 (Table S1 ) was used as the control in the epitope screening experiment.
5
Cell Viability Assay with Inhibitors
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The HEK293T and tumor cell lines were originally obtained from ATCC or JCRB in 2014, where mycoplasma contamination was tested and cell characterization was performed using polymorphic short tandem repeat (STR) profiling. Cells were cultured in RPIM 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Millipore). ABT-263, spautin-1 and other inhibitors were purchased from Selleck Chemicals. All inhibitors were reconstituted in DMSO (Sigma-Aldrich) at a stock concentration of 10 mM. For cell viability assays, cells were seeded at 100,000 cells per well in growth media supplemented with 10% fetal bovine serum in 6-well plates, allowed to adhere overnight, and treated with a serial dilution of inhibitors for 5 days. Cells were fixed with formalin and stained with crystal violet.
6
Culturing MDA-MB-231 Breast Cancer Cells
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The human breast cancer cell line MDA-MB-231 was purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured in RPMI-1640 (GIBCO, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Sijiqing, Hangzhou, China), 100 U/ml penicillin G, and 100 μg/ml streptomycin at 37 °C, 95% relative humidity, and 5% CO2 with 20% oxygen (normoxia), or 1% oxygen (hypoxia). Galactose media was prepared as below: RPIM-1640 deprived of glucose (Invitrogen 11879-020) supplemented with 10 mM galactose, 5 mM HEPES, 10% FBS, 1 mM sodium pyruvate, and pen-strep as above.
7
Culturing Osteoblastic Cell Lines
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Human fetal osteoblastic cell line (hFOB) and OS cell lines (Saos2, MNNG/HOS, U2OS, and MG63) and HEK293T were purchased from the Institute of Cell Bank for Biological Sciences (Shanghai, China). Human osteosarcoma 143B cells (ATCC CRL-8303), suitable transfection host cells, are thymidine kinase deficient (TK-) and resistant to BUdR [9 (link)]. Cells were maintained at 37°C in a humidified air atmosphere containing 95% air and 5% CO2 in DMEM or RPIM-1640 with 10% fetal bovine serum (Invitrogen), 100U/ml penicillin (Sigma-Aldrich) and 100mg/ml streptomycin (Sigma-Aldrich). HFOB was cultured according to ATCC protocols. MEFs were prepared and cultured in DMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1× nonessential amino acids and 0.1 mM 2-mercaptoethanol (Invitrogen) as described before. To remove functional Dicer, MEFs were treated with Adreno-Cre virus, added at a multiplicity of infection (MOI) of ~100 and performed further analysis.
8
Cell Culture of Liver and HSC Lines
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Human normal liver L-02 cells and human HSC lines LX-2 were obtained from Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPIM1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37°C with 5% CO2.
9
Culturing Gastric Cancer Cell Lines
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Four GC cancer cell lines (BGC-823, SGC-7901, HGC-27, and MKN-45) and a human gastric epithelial cell line (GES) were obtained from the Chinese Academy of Science (Shanghai, China). Cells were cultured in 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), Roswell Park Memorial Institute-1640 (RPIM-1640; Invitrogen, Carlsbad, CA, USA), and penicillin/streptomycin (Sigma, St. Louis, MO, USA) in a 5% CO2 incubator at 37°C.
10
HCC Cell Lines and Transfection Protocols
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HCC cell lines SNU475, SMMC-7721, MHCC-97H, Hep3B, HepG2, normal liver cell LO-2, and 293T cells were commercially obtained from the Chinese Academy of Sciences (Beijing, China). Cells were cultured in RPIM-1640 (Invitrogen, USA) with 10% FBS (Gibco, USA) at 37°C in a 5% CO2 atmosphere. All shRNAs, lentiviruses, and plasmids were synthesized and purchased from GeneChem (Shanghai, China). All transfections were completed with Lipofectamine 2000 (Invitrogen, USA).
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