The human ECC cell lines, EGI-1 and TFK-1 (Saijyo et al., 1995 (link)), were purchased from the DSMZ (German Collection of Microorganisms and Cell Cultures, Human and Animal Cell Lines, Germany). EGI-1 cells were cultured in DMEM (Welgene, Korea) containing 10% fetal bovine serum (FBS, Atlas, USA) and MEM amino acids (both essential and non-essential, Gibco Life Technologies, USA). Lenti-X 293T cells (Clontech Laboratories, Inc., USA) were maintained in DMEM supplemented with 10% FBS; TFK-1 cells were cultured in RPMI-1640 media (Welgene), supplemented with 10% FBS. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
Rpmi 1640 media
Manufactured by Welgene
Sourced in United States
RPMI-1640 is a widely used cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides a balanced salt solution, essential amino acids, vitamins, and other nutrients required for cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Welgene (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Welgene (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Welgene (3 mentions)
Mem media, by Welgene (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Streptomycin, by Welgene (2 mentions)
Penicillin, by Welgene (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Welgene (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Hepes buffer, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Mem amino acids, by Thermo Fisher Scientific (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Penicillin and streptomycin, by Welgene (1 mentions)
31 protocols using rpmi 1640 media
1
Cell Culture Protocol for ECC Cell Lines
2
Culturing Human Cancer and Normal Cells
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Human colon cancer cell lines HCT116 and HT-29, human lung cancer cell lines A549 and H460, and human breast cancer cell line MCF-7 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Human normal lung epithelial cell line BEAS-2B and human normal colon fibroblast cell line CCD-18Co was obtained from the ATCC. HCT116, A549, H460, and BEAS-2B cells were maintained in RPMI1640 media (Welgene, Daegu, Korea) supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone, Rockford, IL) and 1% (v/v) penicillin and streptomycin (Invitrogen, San Diego, CA). HT29 and MCF-7 cells were maintained in DMEM media (Welgene) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin and streptomycin. CCD-18Co cells were maintained in MEM media (Welgene) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin and streptomycin.
3
Laser-induced Apoptosis in Regulatory T Cells
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CD4+ T cells were enriched from Foxp3-GFP mouse spleens using a MagniSort Mouse CD4 T-cell Enrichment Kit (eBioscience). Foxp3+ cells were then sorted for using a FACSAria II (BD Biosciences). Sorted cells were stained with 5 μg of anti-CD25-Ce6 or isotype-Ce6 on ice for 25 min. Stained cells were washed and 5 × 104 cells were plated in each well of 96-well flat-bottom plates (FALCON) in RPMI-1640 media (Welgene) with 10% fetal bovine serum (Welgene). Plates were centrifuged for 5 min at 1500 rpm. Cells in each well were irradiated with a 660-nm laser (100 mW/cm2) (Micro Laser Systems) for 5 min. Cell apoptosis after 30 min or 15 h of incubation was measured by detecting surface annexin V (BioLegend) expression by a FACSCalibur (BD Biosciences).
4
Powdered Kamut Sprout Bioactivity Analysis
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Powdered kamut sprout product, used as a raw material, was purchased from Jusung Co. Ltd., Korea. The yeast malt (YM, Lot. 8282839) and Man Rogosa Sharpe (MRS, Lot. 2298709) media that was used for the fermentation were obtained from DIFCO. Food-grade ethanol was used for extraction. The reagents for the β-glucan analysis were obtained by purchasing a β-glucan assay kit (K-YBGL) from Megazyme. Potassium hydroxide and sodium acetate were purchased from DUKSAN, Korea. Folin & Ciocalteu’s phenol reagent (FCR) and gallic acid for the total polyphenol analysis were acquired from Merck, Korea. Sodium carbonate was obtained from Daejung chemicals, Korea. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and L-ascorbic acid for anti-oxidant efficacy testing were purchased from Merck, Korea. FBS, RPMI-1640 media, as well as Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Welgene, Korea, and lipopolysaccharide (LPS), dexamethasone (DEX), and dimethyl sulfoxide (DMSO) were purchased from Merck, Korea. All of the reagents utilized in this research were of extra-pure (analytical) grade.
5
Synthesis and Characterization of mPEG-Ce6 Conjugates
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Methoxy polyethylene glycol amine [mPEG-NH2, molecular weight (Mw) = 5 kDa] was purchased from Laysan Bio Inc. (Alabama, USA). TCPO and sodium cyanoborohydride were purchased from Tokyo Chemical Industry (Tokyo, Japan). CMD sodium salt (Mw = 10 to 20 kDa), N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC•HCl), 4-(dimethyl amino)pyridine (DMAP), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), 2′,7′-dichlorofluorescin diacetate (DCF-DA), TMP, DMPO, and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The Ce6 was obtained from Frontier Scientific Inc. (Logan, Utah, USA). Human colorectal adenocarcinoma cells (HT29) were purchased from the American Type Culture Collection (Rockville, MD, USA). For cell culture, RPMI 1640 media, antibiotic-antimycotic solution, trypsin-EDTA, and Dulbecco’s PBS (DPBS) were purchased from Welgene Inc. (Daegu, Korea). All experiments involving live animals were carried out in accordance with the relevant laws and institutional guidelines of Sungkyunkwan University.
6
Screening Anti-Cancer Stem Cell Agents
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We cultured HCT116, HT29, and DLD-1 cells under standard culture conditions in RPMI-1640 media (Welgene) containing 10% FBS (Atlas Biologicals) and 1 × penicillin-streptomycin (Corning). For the drug treatments, we dissolved JIB-04 (Cayman) and salinomycin (Cayman) in DMSO and diluted the solutions to the appropriate concentrations for the experiments. The following chemicals were used for the primary screening of anti-CSC agents: IOX1 (Cayman), SAHA (Cayman), trichostatin (TSA) (Cayman), EPZ5676 (APEXBIO), eosin Y disodium trihydrate(AMI-5) (Santa Cruz), 2-PCPA(Cayman), sirtinol (Cayman), pargyline (Sigma-Aldrich), paclitaxel (Cayman) 5-FU (Cayman), etoposide (Cayman), nocodazole (Sigma-Aldrich), calmidazolium chloride (Tocris Bioscience), beta-lapachone (Cayman).
7
Culturing Human and Mouse Lung Cancer Cells
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Human A549, H1299, H1993, H292, H358, and H460 cells from ATCC were cultured in RPMI-1640 media (Welgene) and mouse LLC1 cells from ATCC were cultured in DMEM/F12 media (Gibco). PC9 cells, originally provided by Jin Kyung Rho (Asan Medical Center, University of Ulsan, Seoul, Korea), were cultured in RPMI-1640 media. All media contained 10% FBS, 2 mM L-glutamine, 100 IU ml−1 penicillin/streptomycin (Invitrogen). All cells were obtained in 2014, 2015, and 2017 and passaged 4 to 20 times after obtaining. All cells were grown in a humidified incubator at 37 °C with 5% CO2 and were tested regularly for mycoplasma contamination. A549, H1993, and PC9 cells were authenticated at the College of Pharmacy, Seoul National University and LLC1 cells were authenticated at the College of Veterinary Medicine, Seoul National University.
8
Cell Proliferation and Colony Formation
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For cell proliferation assays, 1×103 cells were plated in a 96-well plate and proliferation was measured with the EZ-Cytox Cell Viability Assay kit (Itsbio) using a microplate reader (Molecular Devices, LLC) at 450 nm. For colony-forming assays, 1×103 cells were plated in a 6-well plate. RPMI-1640 Media (Welgene Inc.) were replaced every 3 days then cells were incubated at 37°C. After 2 weeks, colonies were stained with crystal violet solution (0.5 crystal violet, 3.7 formaldehyde, 30.0% ethanol) for 2 h at room temperature, and the number of viable cells was manually counted in each well. All assays were performed in triplicate.
9
Fluorescent Probe Synthesis and Characterization
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Tetraethyl orthosilicate (≥99.0%), 3-aminopropyl triethoxysilane (97%), ammonium hydroxide solution (28.0~30.0%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (commercial grade), N-hydroxysulfosuccinimde sodium salt (sulfo-NHS; ≥98%), copper(II) chloride (99.995%), and Papain (10 units/mg protein) were obtained from Sigma Aldrich (St. Louis, MO, USA). Ethanol and Cy5.5-NHS fluorescent dye were obtained from Duksan (Ansan, Republic of Korea) and Bioacts (Incheon, Republic of Korea), respectively. RPMI-1640 media, fetal bovine serum, penicillin, and streptomycin were purchased from WELGENE Inc. (Daegu, Republic of Korea). DMEM media was commercially purchased from Genedepot (Barker, TX, USA). Hoechst 33342 was purchased from Invitrogen (Thermofisher Scientific; Waltham, MA, USA). The 4T1 (murine breast adenocarcinoma) and HDF (human dermal fibroblast) cells were purchased from ATCC (American Type Culture Collection; Manassas, VA, USA). TEM grid (Carbon Film 200 Mesh copper) was purchased from Electron Microscopy Sciences (Atlanta, GA, USA). Six-week-old female Balb/c nude mice were purchased from OrientBio, Inc. (Seongnam, Republic of Korea). Customized enzyme-cleavable peptides with NIR dye (Cy5.5-Gly-Phe-Leu-Gly-Gly-Lys-Gly-Gly-NHS) were ordered from Peptron (Daejeon, Republic of Korea). All chemicals were used without any purification.
10
C57/BL6 Mouse T Cell Culture
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Eight to 10-week-old female C57/BL6 (OrientBio, Seongnam city, Korea) mice were used. The mice were maintained in a specific pathogen-free facility, and all experiments were approved by Korea University Institutional Animal Care and Use Committee (KUIACUC-2020-0077). T cells were isolated from peripheral lymph nodes and cultured in RPMI 1640 media (WelGENE Inc, Daegu, Korea) with FBS (10 %, WelGENE Inc), penicillin (100 U/mL, Invitrogen, Carlsbad, CA, USA), streptomycin (0.1 mg/mL, Invitrogen), and 2-ME (50 μM, Sigma-Aldrich, St Louis, MO, USA).
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