Rpmi 1640

Manufactured by Genesee Scientific

Sourced in United States

RPMI 1640 is a widely-used cell culture medium designed to support the growth and maintenance of a variety of cell types, including human and animal cells. It provides a balanced salt solution and essential nutrients necessary for cell proliferation and survival.

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RPMI 1640 medium (4 citations) RPMI 1640 media (3 citations)

8 protocols using rpmi 1640

Characterization of Human Cancer Cell Lines

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A549 (CCL-185)- collected from a 58-year old Caucasian male, it is a hypotriploid human cell line with the modal chromosome number 66 which can be found in 24% of cells, NCI-H1299 (CRL-5803)- established from a lymph node of 43-year old White male patient with lung cancer who received prior radiation therapy, MDA-MB-231(HTB-26)- obtained from a 51-year old White female, it is an aneuploid female with a modal chromosome number 64, and MDA-MB-468 (HTB-132)- isolated from a pleural effusion of a 51-year old Black woman with a metastatic breast adenocarcinoma, an aneuploid female with most chromosome counts in the hypertriploid range with a modal chromosome number 64. The A549, MDA-MB-231, and MDA-MB-468 cells were cultured in high glucose Modified Eagle Medium (DMEM) (Genesee Scientific, San Diego, CA, USA) while NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37ºC in 5% CO₂/95% humidified air. In all cases, treatment with experimental compounds was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum.

Tawfeeq N., Lazarte J.M., Jin Y., Gregory M.D, & Lamango N.S. (2023). Polyisoprenylated cysteinyl amide inhibitors deplete singly polyisoprenylated monomeric G-proteins in lung and breast cancer cell lines. Oncotarget, 14, 243-257.

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Cancer Cell Line Culturing and Antibody Verification

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All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231, A549, and MIAPaCa-2 cells were cultured in high glucose Modified Eagle Medium (DMEM, Genesee Scientific, San Diego, CA, USA). NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37 °C in 5% CO₂/95% humidified air. In all cases, treatment with experimental agents was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum. Antibodies specific to B-Raf (Cat. #14814), Phospho-B-Raf (Ser445) (Cat. #2696), c-Raf (Cat. #53745), Phospho-c-Raf (Ser338) (Cat. #9427), MEK1/2 (Cat. #8727), Phospho-MEK1/2 (Ser217/221) (Cat. #9154), p44/42 MAPK (Erk1/2) (Cat. #4695), RSK1/RSK2/RSK3 (Cat. #9355), Phospho-p90RSK (Ser380) (Cat. #11989), GAPDH (HRP Conjugate) (Cat. #8884), α-Actinin (HRP Conjugate) (Cat. #12413), and anti-rabbit IgG, HRP-linked Antibody (Cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Tawfeeq N., Jin Y, & Lamango N.S. (2021). Synthetic Optimization and MAPK Pathway Activation Anticancer Mechanism of Polyisoprenylated Cysteinyl Amide Inhibitors. Cancers, 13(22), 5757.

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Culturing Cisplatin-Resistant Ovarian Cancer Cells

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F2 and Ovcar8 cell lines were grown in RPMI 1640 (10% fetal bovine serum and 1% penicillin/streptomycin) (Genesee Scientific) in a tissue culture incubator at 37°C, 5% CO₂, and 90% humidity. Cells were passaged every 2-4 days using 0.25% trypsin (Genesee Scientific). Where indicated, cells were transfected with Lipofectamine 2000 (Thermo Fisher). F2 cells are cisplatin resistant cells taken from primary patient tissue with high grade serous ovarian cancer and were a gift from Dr. Gil Mor at Yale University.

Shahin S.A., Wang R., Simargi S.I., Contreras A., Parra L., Qu L., Wen W., Dellinger T., Unternaehrer J., Tamanoi F., Zink J.I, & Glackin C.A. (2018). Hyaluronic acid conjugated nanoparticle delivery of siRNA against TWIST reduces tumor burden and enhances sensitivity to cisplatin in ovarian cancer. Nanomedicine : nanotechnology, biology, and medicine, 14(4), 1381-1394.

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Culturing Cisplatin-Resistant Ovarian Cancer Cells

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A2780R and all derivatives of Ovcar8 were grown in RPMI 1640 (Genesee Scientific, San Diego, CA) in a tissue culture incubator at 37°C, 5% CO₂, and 90% humidity. Growth medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were passaged every 2–4 days using 0.25% trypsin (Genesee Scientific). Where indicated, cells were transfected with Lipofectamine 2000 (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions. A2780R cells are a cisplatin resistant derivative of A2780 and were a gift from Dr. John Yim’s lab at City of Hope.

Roberts C.M., Shahin S.A., Wen W., Finlay J.B., Dong J., Wang R., Dellinger T.H., Zink J.I., Tamanoi F, & Glackin C.A. (2016). Nanoparticle delivery of siRNA against TWIST to reduce drug resistance and tumor growth in ovarian cancer models. Nanomedicine : nanotechnology, biology, and medicine, 13(3), 965-976.

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Characterization of Breast Cancer Cell Lines

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The characteristics of the human breast cancer cell lines described by Smith et al. [39 (link)] and used for the cytotoxicity assays are shown in Table 1. The human embryonic kidney cell line HEK293 and breast cancer cell lines BT-20, MCF-7, MDA-MB-231, MDA-MB-436, and ZR-75-1 were obtained from ATCC (Manassas, VA) and maintained in DMEM supplemented with 10% FBS (Gemini, West Sacramento, CA), 1% penicillin/streptomycin (Thermo Fisher, Waltham, MA) and 1% NEAA (Thermo Fisher). The human mammary gland epithelial cell line, MCF-12A, was obtained from ATCC and maintained in DMEM/F12 containing 5% donor horse serum, 0.5 μg/mL hydrocortisone, 0.01 mg/mL bovine insulin, 100 ng/mL cholera toxin, and 20 ng/mL human EGF. The Chinese hamster ovary cell lines, CHO and CHO-CAR (stably expressing the CXADR or CAR cDNA) as characterized previously [40 (link)], were kindly provided by Rhonda Cardin (Louisiana State University School of Veterinary Medicine, Baton Rouge, LA) and maintained in RPMI 1640 (Genesee Scientific, San Diego, CA) containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, 10 μg/mL thymidine (Sigma-Aldrich, St. Louis, MO), 10 μg/mL adenosine (Sigma-Aldrich) and 10 μg/mL 2-deoxyadenosine (Sigma-Aldrich). CHO-CAR cells were also supplemented with 100 μg/mL Zeocin (InvivoGen, San Diego, CA). All cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere.

O’Bryan S.M, & Mathis J.M. (2021). CXCL12 Retargeting of an Oncolytic Adenovirus Vector to the Chemokine CXCR4 and CXCR7 Receptors in Breast Cancer. Journal of cancer therapy, 12(6), 311-336.

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Breast Cancer Cell Line Cultivation and Treatment

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Human breast cancer cell lines used in experiments were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Human luminal A MCF7 and T47D [ER+, PR+, HER2−] and triple negative basal B MDA-MB-231 [ER−, PR−, HER2− were cultured in RPMI 1640 (Genesee Scientific; San Diego, CA), while BT474 [ER+, PR+, HER2+] and triple negative basal A MDA-MB-468 [ER−, PR−, HER2−] cells were cultured in DMEM (Genesee Scientific). Sk-Br-3 [ER−, PR−, HER2+] breast cancer cells were cultured using McCoy’s 5A media (ATCC). All cell media contained 10% v/v Fetal Clone III (Thermo Fisher; Waltham, MA) and 1% v/v penicillin/streptomycin (Genesee Scientific). Cells were cultivated in tissue culture treated T-75 flasks (Genesee Scientific) kept in a Model 3110 (Forma Scientific; Marietta, OH) incubator at 37 °C and 5% CO₂. Cells grown to ~ 75% confluence before plating for experiments. Cells were treated with recombinant human OSM, IL-6, leukemia inhibitory factor (LIF) (25 ng/mL), and/or interleukin 1β (IL-1β; 10 ng/mL) from Peprotech Inc. (Rocky Hill, NJ) at various time intervals depending on the experiment and highlighted in the figures.

Dinca S.C., Greiner D., Weidenfeld K., Bond L., Barkan D, & Jorcyk C.L. (2021). Novel mechanism for OSM-promoted extracellular matrix remodeling in breast cancer: LOXL2 upregulation and subsequent ECM alignment. Breast Cancer Research : BCR, 23, 56.

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One-step Growth Curve for Virus Infection

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One or two days prior to infection, cells were seeded in a 24-well plate and infected at 60–80% confluency. Cells were infected in triplicate at a MOI of 0.01 for all cells except Aag2, WT 3T3, and 3T3 Mxra8 KO cells, which were infected at MOI 0.1. Virus was diluted in RPMI-1640 (Genesee Scientific) media containing 2% FBS and 10 mM HEPES. One hour post-infection, viral inoculum was removed, cells were washed once with PBS, and the appropriate culture media was added to the cells. Cells were incubated at either 37°C or 28°C, according to cell type and conditions listed above. Supernatant was collected every 24 hours for the indicated time points and frozen until viral titration. One-step growth curves were performed by infecting Aag2 cells at a MOI of 10. Cells were chilled at 4°C for 30 minutes and adsorption was performed at 4°C for 30 minutes to synchronize infection. Unbound virus was washed away with cold PBS six times, and internalization and infection were initiated by adding media to cells and incubating at 28°C. Supernatants were harvested every 3 hours post-infection, and all infectious virus was quantified by plaque assay.

Cereghino C., Roesch F., Carrau L., Hardy A., Ribeiro-Filho H.V., Henrion-Lacritick A., Koh C., Marano J.M., Bates T.A., Rai P., Chuong C., Akter S., Vallet T., Blanc H., Elliott T.J., Brown A.M., Michalak P., LeRoith T., Bloom J.D., Marques R.E., Saleh M.C., Vignuzzi M, & Weger-Lucarelli J. (2023). The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito. PLOS Pathogens, 19(4), e1010491.

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Lymphocyte Stimulation and Cytokine Analysis

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Single cell suspensions from aseptically removed the head and neck lymph nodes (HNLNs), mesenteric LNs (MLNs), and spleens were prepared as previously described [35 (link)]. Lymphocytes were cultured in a complete medium: RPMI 1640 with 2 mM l-glutamine (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Atlanta Biologicals, Oakwood, Georgia), plus supplements (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. Lymphocytes were cultured at 10⁶ cells/well in 96-well, round-bottomed tissue culture plates (Millipore, Billerica, MA) coated with 5 μg/mL anti-CD3 mAb (clone 17A2; Invitrogen, Carlsbad, CA, USA) plus 2.5 μg/mL of soluble anti-CD28 mAb (clone 37.51; Invitrogen) was stimulated for 48 h at 37 °C. Lymphocytes were stimulated in triplicate for 2 days for flow cytometry analysis or for 4 days for collection of cell culture supernatants, which were then stored at − 20 °C until assayed by cytokine-specific ELISAs.

Akgul A., Maddaloni M., Jun S.M., Nelson A.S., Odreman V.A., Hoffman C., Bhagyaraj E., Voigt A., Abbott J.R., Nguyen C.Q, & Pascual D.W. (2021). Stimulation of regulatory T cells with Lactococcus lactis expressing enterotoxigenic E. coli colonization factor antigen 1 retains salivary flow in a genetic model of Sjögren’s syndrome. Arthritis Research & Therapy, 23, 99.

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