Rpmi medium

Manufactured by Thermo Fisher Scientific

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RPMI medium is a commonly used cell culture medium formulation developed at the Roswell Park Memorial Institute. It provides a balanced salt solution and essential nutrients to support the growth and maintenance of a variety of cell types, including mammalian cells, in in vitro cell culture applications.

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1 417 protocols using rpmi medium

Cell Culture Conditions for Diverse Cell Lines

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Murine T-lymphoma ^MYC-Tet-Off cells (female) were grown in RPMI medium (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma), 1% penicillin/streptomycin solution (Sigma), 1% glutamine solution (Thermo Fisher Scientific), 1% MEM non-essential amino acids (Thermo Fisher Scientific), and 50 μM β-mercaptoethanol (Sigma). Human U2OS (female), U2OS ^MYC-Tet-On, HEK293 (female) and HeLa (female) cells, as well as murine NIH 3T3 (not applicable) cells, were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin solution (Sigma). Human mammary epithelial cells (HMLE, female) were cultivated in advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with 20 ng/ml EGF (human) (Thermo Fisher Scientific), 0.5 μg/ml Hydrocortisone (Sigma), 10 μg/ml Insulin (human) (Sigma), 1% penicillin/streptomycin solution (Sigma), 1% glutamine solution (Thermo Fisher Scientific) and 15 mM HEPES (pH 7.2). Human Neuroblastoma SH-EP cells (female) were verified by STR profiling and grown in RPMI medium (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin solution. All cell lines were cultured at 37°C, 5% CO₂ and routinely screened and found negative for mycoplasma contamination in a PCR-based assay.

Baluapuri A., Hofstetter J., Dudvarski Stankovic N., Endres T., Bhandare P., Vos S.M., Adhikari B., Schwarz J.D., Narain A., Vogt M., Wang S.Y., Düster R., Jung L.A., Vanselow J.T., Wiegering A., Geyer M., Maric H.M., Gallant P., Walz S., Schlosser A., Cramer P., Eilers M, & Wolf E. (2019). MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation. Molecular Cell, 74(4), 674-687.e11.

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Macrophage differentiation and SIMV dose determination

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The monocyte cell line THP-1 (ATCC^® TIB-202™) derived from human peripheral blood from a one year old male was used to obtain macrophages to determine effective dose ranges. THP-1 cells were expanded in ultra-low attachment flasks (Corning, USA, cat# CLS3814) in RPMI medium (Thermo Fisher Scientific, MA, USA) with 10% heat inactivated fetal bovine serum (FBS), and 100 U/ml penicillin and 100 μg/ml streptomycin sulfate. THP-1 at 0.6 million cells/ml were differentiated to macrophages through incubation in 100 mM phorbol-12-myristate 13-acetate (PMA) (Sigma, USA, P8139) for 24 hrs. Macrophages were then gently scraped off the ultra-low attachment culture flask and seeded at 1 ×106 cell/ml on bCaP coated disks with or without the drugs/cytokines that had been incubated for 30 min in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific, MA, USA) prior to cell culture. In the SIMV dose determination studies, cells were cultured with 100ng/ml of both LPS and IFNγ in the medium to provide the M1 stimulation, and then refreshed on day 3 with drug free medium to evaluate the M2 effect of simvastatin.

Alhamdi J.R., Peng T., Al-Naggar I.M., Hawley K.L., Spiller K.L, & Kuhn L.T. (2018). Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings. Biomaterials, 196, 90-99.

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Culturing Human Cell Lines

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Human esophageal squamous epithelial cancer cell line TE-7 was kindly provided by Dr. Hainault (IARC, Lyon, France). These cells were maintained in RPMI medium (Invitrogen) supplemented with 5% FBS (Atlanta Biologicals) and 1% penicillin/streptomycin (Invitrogen). HEK 293T cells and L929 cells were grown in DMEM medium (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals) and 1% penicillin/streptomycin (Invitrogen). Jurkat cells were cultured in RPMI medium (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals) and 1% penicillin/streptomycin (Invitrogen).

Caldwell J.M., Collins M.H., Kemme K.A., Sherrill J.D., Wen T., Rochman M., Stucke E.M., Amin L., Tai H., Putnam P.E., Jiménez-Dalmaroni M.J., Wormald M.R., Porollo A., Abonia J.P, & Rothenberg M. (2017). Cadherin 26 is an alpha integrin-binding epithelial receptor regulated during allergic inflammation. Mucosal immunology, 10(5), 1190-1201.

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Culturing Human Cell Lines

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Culturing Human Renal and Murine Colorectal Carcinoma Cells

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The human renal cell carcinoma cell line, SKRC-52, was kindly provided by Professor E. Oosterwijk (Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands). SKRC-52 cells were cultured in RPMI Medium (Invitrogen), supplemented with FCS (10%, Invitrogen) and Antibiotic-Antimycotic (1%, Invitrogen), at 37 C, 5% CO 2 . Cells at 90%-100% confluence were detached using 0.05% Trypsin-EDTA (Invitrogen) and reseeded at a dilution of 1:6.
The murine colorectal carcinoma CT26-3E10-transfected cells (31) were cultured in RPMI Medium (Invitrogen), supplemented with FCS (10%, Invitrogen) and Antibiotic-Antimycotic (1%, Invitrogen), at 37 C, 5% CO 2 . Cells at 90%-100% confluence were detached using 0.05% Trypsin-EDTA (Invitrogen) and reseeded at a dilution of 1:6.

Millul J., Krudewig C., Zana A., Dakhel Plaza S., Puca E., Villa A., Neri D, & Cazzamalli S. (2021). Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX. Molecular cancer therapeutics, 20(3).

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Silencing UBE2C in ESCC Cell Lines

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TE-1 and TE-13 cell lines were derived from ESCC and were kindly provided by Dr. Pierre Hainaut (IARC, France). Both lineages were cultured in RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% of the cocktail penicillin / glutamine / streptomycin (Invitrogen) and maintained at 37°C under 5% CO₂. For silencing experiments, 1.5×10⁵ cells were seeded in a 6-well plate. 24 hours prior siRNA transfection, TE-1 and TE-13 cells were treated with Thymidine (Sigma) at the concentration of 4 mM in RPMI medium supplemented with 0.5% FBS (Gibco), in order to synchronize them in G1/S phase of the cell cycle. Then, double-strand siRNA oligonucleotides targeting UBE2C gene or fluorescent scrambled control siRNA (#1022563) were transfected into TE-1 and TE-13 cells at a final concentration of 120 nM. All siRNA duplexes were purchased from Qiagen (Qiagen, Valencia, CA) and were transfected by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's recommendations.

Palumbo A J.r., Costa N.M., De Martino M., Sepe R., Pellecchia S., Leite de Sousa V.P., Neto P.N., Kruel C.D., Bergman A., Nasciutti L.E., Fusco A, & Pinto L.F. (2016). UBE2C is overexpressed in ESCC tissues and its abrogation attenuates the malignant phenotype of ESCC cell lines. Oncotarget, 7(40), 65876-65887.

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Cell Culture Protocols for Rare Cancer Research

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The NCC-CDS1-X1(CDS1) and NCC-CDS2-C1(CDS2) cell lines were kindly provided by Tadashi Kondo (Division of rare cancer research head, National Cancer Center Research Institute, Tokyo, Japan). HEK293T cells were obtained from the ATCC. CDS1 and CDS2 cells were grown in RPMI medium (Gibco, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (PenStrep, Gibco) in adherent cell culture conditions. Cells were passed in RPMI medium (Gibco) supplemented with 20% KO serum (Gibco) one week prior experimentations. LentiX HEK293T, HeLa, and RDES cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% PenStrep. All cells were cultured at 37 °C with 5% CO₂. Cells were maintained and split every 2–4 days (at approximately 80% confluence). Cells were cryopreserved in cryogenic medium [90% FBS with 10% dimethyl sulfoxide (DMSO)] into liquid nitrogen for long-term storage. RD-ES Ewing’s sarcoma cell line was obtained from ATCC, cells were cultured in RPMI medium supplemented with 15% FBS and 1% PenStrep and sub-cultured twice per week.

Bakaric A., Cironi L., Praz V., Sanalkumar R., Broye L.C., Favre-Bulle K., Letovanec I., Digklia A., Renella R., Stamenkovic I., Ott C.J., Nakamura T., Antonescu C.R., Rivera M.N, & Riggi N. (2024). CIC-DUX4 Chromatin Profiling Reveals New Epigenetic Dependencies and Actionable Therapeutic Targets in CIC-Rearranged Sarcomas. Cancers, 16(2), 457.

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Macrophage-like cell lines for research

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HD11 cells (chicken macrophage-like cell line) were derived from chicken bone marrow macrophages by transforming the avian leukemia virus (34 (link)). HD11 macrophages were kindly provided by Shaohui Wang (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences) and cultured as the previously described (15 (link), 35 (link)). HD11 cells were cultured in RPMI 1640 media (Gibco) (at a humidified incubator for 41.5°C and 5% CO₂), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine (Gibco), 1 mM of sodium pyruvate 0.1 mM of NEAA, and 10 mM HEPES. U937 cells (human monocytic lymphoid cell line) was originally obtained from the ATCC and cultured in RPMI medium (Gibco) as the previously described (36 (link)). U937 cells were maintained in RPMI medium (at a humidified incubator for 37.5°C and 5% CO₂), supplemented with 10% FBS (Gibco) and 2 mM l-glutamine. To obtain a macrophage-like phenotype, U937 cells were differentiated with 25 ng/ml phorbol 12-myristate 13-acetate for 24 h.

Zhuge X., Sun Y., Xue F., Tang F., Ren J., Li D., Wang J., Jiang M, & Dai J. (2018). A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages. Frontiers in Immunology, 9, 788.

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Cell Lines for Viral Infection Studies

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The RMCE-HILO HEK293T acceptor cells were obtained from Eugene V. Makeyev (Nanyang Technological University, Singapore) and maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL) with 10% FetalPlex Serum Complex (Gemini Bio) and penicillin and streptomycin (50 U/ml). Ramos cells (uninfected Burkitt Lymphoma from ATCC CRL-1596) and BJAB (uninfected B cell lymphoma) cells were obtained from ATCC and maintained in RPMI medium (Gibco BRL) containing 10% heat-inactivated fetal bovine serum and penicillin and streptomycin (50 U/ml). KSHV⁺ single positive PEL cells (BCBL1 and BC3) and double positive KSHV and EBV infected PEL cells (BC1) were provided by Yan Yuan (University of Pennsylvania) and maintained in RPMI medium (Gibco BRL) containing 10% heat-inactivated fetal bovine serum and penicillin and streptomycin (50 U/ml).

Calderon A., Soldan S.S., De Leo A., Deng Z., Frase D.M., Anderson E.M., Zhang Y., Vladimirova O., Lu F., Leung J.C., Murphy M.E, & Lieberman P.M. (2020). Identification of Mubritinib (TAK 165) as an inhibitor of KSHV driven primary effusion lymphoma via disruption of mitochondrial OXPHOS metabolism. Oncotarget, 11(46), 4224-4242.

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Isolation of Microglia from Mouse Brain

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Mice were decapitated, and brains were homogenized in RPMI medium (21875-034, Gibco) with a 15 ml Dounce homogenizer on ice until the sample is fully homogeneous, without any visible tissue fragments. The final homogenate was filtered through a 70 μm cell strainer (431751, Corning). Following 5 min centrifugation at 380 g at 4°C, cell pellets were resuspended with 7 ml RPMI medium (11875093, Gibco) and mixed with 3 ml stock isotonic Percoll (SIP) solution which was made by mixing one part 10x HBSS (14185052, Gibco) in nine parts of Percoll plus (GE17-5445-01, Sigma-Aldrich). The cell suspension was then layered slowly on top of 2 ml of 70% Percoll solution which was prepared by mixing three parts of HBSS (14170112, Gibco) with seven parts SIP in a new 15 ml falcon and centrifuged at 500 g speed for 30 min at 18°C, with minimal acceleration and break rate. After centrifugation, the fuse interphases were transferred into a new 15 ml falcon with 8 ml HBSS and centrifuged at 500 g for 7 min again. The supernatant and cell debris were discarded and microglial cells were collected for RNA isolation.

Wang X.L., Wolff S.E., Korpel N., Milanova I., Sandu C., Rensen P.C., Kooijman S., Cassel J.C., Kalsbeek A., Boutillier A.L, & Yi C.X. (2020). Deficiency of the Circadian Clock Gene Bmal1 Reduces Microglial Immunometabolism. Frontiers in Immunology, 11, 586399.

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