Rpmi 1640 medium

Manufactured by Takara Bio

Sourced in United States

RPMI 1640 medium is a cell culture medium designed to support the growth of a wide range of cell types, including human and animal cells. It is a chemically defined, serum-free medium that provides a balanced formulation of essential nutrients, vitamins, and salts required for cellular proliferation and maintenance.

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RPMI 1640 (8 citations)

10 protocols using rpmi 1640 medium

Establishment and Maintenance of NPC Cell Lines

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Primary NPC fresh tumor tissues and fresh non-cancer nasopharyngitis tissues were acquired from the Tumor Tissue Bank of Sun Yat-Sen University Cancer Center, and the detailed relevant information has been described previously.^{32 (link)} This study was approved by the Institute Research Medical Ethics Committee of Sun Yat-Sen University Cancer Center, and written informed consent was obtained from all patients. NPC cell lines were maintained in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO₂ incubator at 37 °C. Primary NPEC1 cultures and immortalized NPEC1 cells induced by Bmi-1 were established as described previously^{51 (link)} and grown in keratinocyte/serum-free medium (Invitrogen, Carlsbad, CA, USA). Tet-Off-inducible advanced cell lines were maintained in RPMI 1640 medium supplemented with 10% Tet-System Approved FBS (Clontech, Mountain View, CA, USA) with both G418 (400 μg/ml) and doxycycline (200 ng/ml). Tet-Off-inducible RBM24 stable cells were maintained in RPMI 1640 medium supplemented with 10% Tet-System Approved FBS (Clontech) with G418 (400 μg/ml), puromycin (0.5 μg/ml) and doxycycline (200 ng/ml).

Hua W.F., Zhong Q., Xia T.L., Chen Q., Zhang M.Y., Zhou A.J., Tu Z.W., Qu C., Li M.Z., Xia Y.F., Wang H.Y., Xie D., Claret F.X., Song E.W, & Zeng M.S. (2016). RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma. Cell Death & Disease, 7(9), e2352-.

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Establishment and Characterization of Primary Pancreatic Tumor Cells

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Establishment of primary pancreatic tumor cells was performed as described previously⁶⁴. Cells were maintained in RPMI-1640 medium containing 10% FBS (Clontech). All experiments were performed within eight passages to avoid possible gross genomic changes during long-term in vitro culture. Cells were verified by epithelial morphology in two dimensions (2D), expression of the epithelial marker cytokeratin 19 and the ability to form tumors in vivo. Cells were tested for mycoplasma contamination using commercially available kit (PCR-Mycoplasma Test Kit I/C (Promokine PK-CA91-1024) according to manufacturer’s manual at the onset of the work (tested negative) and have never exhibited contamination symptoms after the test. Derivative cells were transduced in vitro with a lentiviral vector-encoding firefly Luciferase and mApple fluorescent protein for combined bioluminescent imaging and flow sorting. To ensure that all cell lines expressed similar levels of luciferase, mApple expressing cells were flow sorted prior to injection into animals.

Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.

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Cell Culture Protocol for Cell Lines

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Jurkat (ATCC, Manassas, VA, USA), Jurkat NFAT (BPS Biosciences, San Diego, CA, USA), THP-1 NF_KB (BPS Biosciences), and U937 (ATCC) cell lines were maintained in RPMI 1640 medium (Clontech, San Jose, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA). RBL2H3 (ATCC) and HEK293 (ATCC) cell lines were maintained in DMEM medium (Clontech) supplemented with 10% fetal bovine serum. The Luva cell line (Kerafast, Boston, MA, USA) was maintained in StemPro-34 SFM medium supplemented with StemPro-34 Nutrient supplement and 2 m m L-glutamine (Gibco). All cells were cultured at 37°C, 5% CO₂, and 95% humidity.

Faouzi M., Wakano C., Monteilh-Zoller M.K., Neupane R.P., Starkus J.G., Neupane J.B., Cullen A.J., Johnson B.E., Fleig A, & Penner R. (2022). Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry. Function, 3(4), zqac033.

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Culturing Cell Lines and PBMCs

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Jurkat, U937, HL-60 cell lines and PBMCs were maintained in RPMI 1640 medium (Clontech) supplemented with 10% fetal bovine serum (FBS). RBL-2H3 cells (WT and M1) were cultured in DMEM medium (Clontech) with 10% fetal bovine serum (FBS). All cells were cultured at 37 °C, 5% CO₂, and 95% humidity.

Faouzi M., Neupane R.P., Yang J., Williams P, & Penner R. (2018). Areca nut extracts mobilize calcium and release pro-inflammatory cytokines from various immune cells. Scientific Reports, 8, 1075.

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c-MYC Induction and Inhibition Assay

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For c-MYC induction experiments, the conditional Tet-MYC vector in P493-6 cells was repressed with 0.1 μg/ml tetracycline (Sigma) for 72 h (31 (link)). P493-6 cells were kindly provided by Chi Van Dang, University of Pennsylvania. MYC expression was induced for 1 h or 24 h by washing the cells three times with RPMI-1640 medium containing 10% tetracycline system-approved FBS (Clontech). Immortalized mammary epithelial cells (IMECs) and MCF10A cells were cultured in DMEM:F12 50:50 medium supplemented with 10 ng/mL EGF, 5 ng/mL insulin, 0.5 μg/mL hydrocortisone, and 5% FBS as previously described (32 ). MCF7, HeLa, HEK293T, and MDA-MB-231 cells were cultured in DMEM/10% FBS. MM1.S cells were propagated in RPMI-1640 medium containing 15% FBS. For SNS-032 and THZ1 (APExBIO) treatments, MYC expression was repressed in P493-6 cells for 72 h and restored through tetracycline withdrawal for 0 or 23 h prior to treatment. Cells were treated with 100 nM SNS-032 and 50 nM or 250 nM THZ1 for 24 h and 1 h, respectively, followed by F buffer (10 mM Tris pH 7.05, 50 mM NaCl, 30 mM Na₄P₂O₇, 5 μM ZnCl₂, 50 mM NaF, 10% Glycerol, 0.5% Triton X-100) lysis for immunoblotting or TRIzol RNA extraction for mRNA analysis. Western blots were imaged using the BioRad Molecular Imager ChemiDoc XRS+ System and band intensity was quantified using ImageJ.

Posternak V., Ung M.H., Cheng C, & Cole M.D. (2016). MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase. Molecular cancer research : MCR, 15(2), 213-224.

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Establishment and Characterization of Primary Pancreatic Tumor Cells

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Tetracycline-Inducible LANA Expression in BJAB Cells

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293T and Vero cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Corning). Most suspension cells (BCBL-1, BC-1, BC3, and BJAB) and tetracycline-inducible TREx/BJAB cells [57 (link)] were maintained in RPMI 1640 medium supplemented with 1% P/S and 10% FBS or 10% Tet system approved FBS (TaKaRa), respectively. Transient transfection was performed using PEI (Sigma) or FuGENE HD (Promega) according to the manufacturers’ instructions. To establish BJAB cells expressing wild-type (WT) or mutant LANA in a tetracycline-inducible manner, pcDNA/FRT/To-LANA/Au or pcDNA/FRT/To-LANA-P1/Au was transfected together with the Flp recombinase expression vector pOG44. Twenty-four hours after transfection, cells were selected using 200 μg/ml of hygromycin B (Invitrogen). The detailed procedure was delineated previously [58 (link)].

Kim Y.J., Kim Y., Kumar A., Kim C.W., Toth Z., Cho N.H, & Lee H.R. (2021). Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen dysregulates expression of MCL-1 by targeting FBW7. PLoS Pathogens, 17(1), e1009179.

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Inducible CD19-targeted CAR T Cells

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Parental NALM-6 (CD19⁺) cells were transduced with pLVX.CD19BBz.mCherry lentivirus, treated with 1 μg/ml doxycycline (Takara Bio USA, 631311), and sorted 48 h following doxycycline treatment on the basis of mCherry expression (>99% purity)^{24 (link)}. This cell line was maintained in RPMI-1640 medium supplemented with tetracycline-free FBS (Takara Bio USA, 631101). All experiments involving this cell line were performed 48 h following doxycycline treatment (1 μg/ml).

Ruella M., Xu J., Barrett D.M., Fraietta J.A., Reich T.J., Ambrose D.E., Klichinsky M., Shestova O., Patel P.R., Kulikovskaya I., Nazimuddin F., Bhoj V.G., Orlando E.J., Fry T.J., Bitter H., Maude S.L., Levine B.L., Nobles C.L., Bushman F.D., Young R.M., Scholler J., Gill S.I., June C.H., Grupp S.A., Lacey S.F, & Melenhorst J.J. (2018). Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nature medicine, 24(10), 1499-1503.

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Prostate Cancer Cell Lines Culture

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All cell lines used in this study were purchased from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, P.R. China). The cell lines included the DU-145, LNCap, and PC-3 human prostate cancer cell lines, as well as the RWPE-1 normal prostate epithelium cell line. All cell lines were cultured in RPMI-1640 medium (Takara Biotechnology Co., Ltd., Dalian, P.R. China) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bovine pituitary extract (0.05 mg/ml; Thermo Fisher Scientific, Inc.) and human recombinant epidermal growth factor (5 ng/ml; Thermo Fisher Scientific, Inc.) were added to the culture medium of the RWPE-1 cells. The cell culture environment was thermostatic at 37°C with constant humidity and 5% CO₂. The cells were mainly seeded into six-well plates at a density of 4 × 10⁵ cells; a lower density was used depending on certain experiments. Once the cells reached 60–70% confluency, all transfections were performed with Invitrogen Lipofectamine® 2000 Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions, with the synthesized RNA mimic or negative control (NC). Transfected cells were cultured for 48 or 72 h at the same conditions described above.

Huang F., Zhao H., Du Z, & Jiang H. (2019). miR-615 Inhibits Prostate Cancer Cell Proliferation and Invasion by Directly Targeting Cyclin D2. Oncology Research, 27(3), 293-299.

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Culturing the INS-1 Rat Insulinoma Cell Line

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The rat insulinoma cell line INS-1 was obtained from the China Center for Type Culture Collection (Wuhan University, Wuhan, China). RPMI-1640 cell culture medium and fetal bovine serum (FBS) were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA).
INS-1 cells were maintained in RPMI-1640 medium containing 11 mM glucose supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 µM β-mercaptoethanol (all Takara Biotechnology Co., Ltd., Dalian, China). Culture medium was replaced daily until cell confluency reached 80–90%.

Yang Y, & Gong L. (2017). Palmitoleate inhibits insulin transcription by activating the ERK1/2 pathway in rat pancreatic β-cells. Experimental and Therapeutic Medicine, 13(6), 2805-2811.

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