Rpmi 1640

Manufactured by Harvard Bioscience

Sourced in Germany, United Kingdom, United States

RPMI 1640 is a cell culture medium commonly used for the growth and maintenance of various cell types, including human and animal cells. It provides a balanced salt solution and essential nutrients required for cell growth and proliferation.

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135 protocols using rpmi 1640

Cell Culture of Cancer Cell Lines

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The osteolytic prostate cancer cell line PC-3 (ATCC CRL1435) was grown in DMEM and the prostate epithelial cell line Ep156T (kindly donated by V. Rotter, Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel) in modified MCDB-153 medium (WKS Diagnostics, Frankfurt am Main, Germany) supplemented with 5 ng/ml epidermal growth factor (Invitrogen), 10 nM R1881 and 50 ug/ml bovine pituitary extract (Gibco). The breast cancer cell line MDA-MB231 (ATCC-HTB-26) was grown in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented with sodium pyruvate. The mouse breast cancer cell line 4T1.2 (kindly donated by CR. Anderson, University of Melbourne, Melbourne) was grown in RPMI 1640 (Biochrom AG, Berlin, Germany). The human osteoinductive prostate cancer cell line VCaP (kindly donated by K. Pienta, University of Michigan, Ann Arbor) was grown in RPMI 1640 medium (Biochrom AG, Berlin, Germany).
All media were supplemented with 10% heat-inactivated fetal bovine serum gold (PAA, The Cell Culture Company).

Hensel J., Wetterwald A., Temanni R., Keller I., Riether C., van der Pluijm G., Cecchini M.G, & Thalmann G.N. (2018). Osteolytic cancer cells induce vascular/axon guidance processes in the bone/bone marrow stroma. Oncotarget, 9(48), 28877-28896.

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Isolation and Culture of Tumor Cells

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The tumor tissue resected from 5 patients who underwent surgery was immediately collected in sterile Phosphate-Buffer saline (PBS) without Ca²⁺ and Mg⁺ (L1825 Biochrom, Berlin, Germany). The tissue was washed (3×) with sterile PBS in order to remove any tissue debris and blood. Afterwards, the tissue was cut in small pieces with a sterile scalpel (Feather, Osaka, Japan). The small pieces were rinsed through a cell strainer (352350 BD Labware, Franklin Lakes, NJ, USA) and washed with Roswell Park Memorial Institute 1640 (RPMI1640) Medium (FG1215 Biochrom, Berlin, Germany). The cell suspension was centrifuged at 1500 rpm for 8 min at room temperature. The pellet was suspended with complete growth medium RPMI 1640 (Biochrom) supplemented with 10% fetal bovine serum (FBS, Biochrom) and 10 U/mL penicillin and 100 µg/mL streptomycin (Biochrom) and the suspension was pipetted in a cell culture 6-well plate (83.3920 Sarstedt, Nümbrecht, Germany). After 2 h, the adherence of the cells was checked. The medium was changed regularly every second day. The cells were then trypsinized and transferred into 25 cm² flasks.

Wächter S., Wunderlich A., Roth S., Mintziras I., Maurer E., Hoffmann S., Verburg F.A., Fellinger S.A., Holzer K., Bartsch D.K, & Di Fazio P. (2018). Individualised Multimodal Treatment Strategies for Anaplastic and Poorly Differentiated Thyroid Cancer. Journal of Clinical Medicine, 7(5), 115.

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Cell Culture Protocol for Tumor and Transduced Cell Lines

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Tumor cell lines, U-251 MG and EGFRvIII-expressing U-251 MG (U-251 MG/EGFRvIII), were described previously (16) . Both cell lines were cultured in DMEM (Gibco) containing 10% FBS (Invitrogen) and 100 U/mL penicillin/streptomycin (Gibco). Tumor cell lines, U-87 MG/EGFRvIII and GOS-3/EGFRvIII, were generated by transduction of U-87 MG tumor cells (ATCC) and GOS-3 [German Collection of Microorganisms and Cell Culture (DSMZ)] with human EGFRvIII cDNA, and were cultured in RPMI1640 (Biochrom) and DMEM containing 10% FBS, 100 U/mL penicillin/streptomycin, and 5 mg/mL Blasticidin (Gibco). The tumor cell line, DK-MG (DSMZ), was cultured in RPMI1640 containing 10% FBS, 100 U/mL penicillin/ streptomycin, and 1 Â nonessential amino acids (NEAA, Biochrom). CHO cells were transduced with wild-type EGFR (HER1), HER2, HER3, HER4, and human and cynomolgus monkey EGFRvIII. CHO cells were cultured in DMEM containing 10% FBS, 10 mmol/L sodium hypoxanthine, 1.6 mmol/L thymidine (1 Â HT Supplement, Gibco), and 1 Â NEAA (Biochrom). Transduced CHO cells were cultured in DMEM containing 10% FBS, 2 mmol/L L-Glutamine (Biochrom), 100 U/mL penicillin/streptomycin, and 1 Â NEAA. T-cell lines, HPB-ALL (DSMZ) and LnPx4119 (described previously; ref. 15) , were cultured in RPMI1640 containing 10% FBS and 100 U/mL penicillin/ streptomycin. All cells were cultured at 37 C in a 5% CO 2 chamber.

Sternjak A., Lee F., Thomas O., Balazs M., Wahl J., Lorenczewski G., Ullrich I., Muenz M., Rattel B., Bailis J.M, & Friedrich M. (2021). Preclinical Assessment of AMG 596, a Bispecific T-cell Engager (BiTE) Immunotherapy Targeting the Tumor-specific Antigen EGFRvIII. Molecular cancer therapeutics, 20(5).

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Culturing Isogenic Human Monocytic Cells

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Isogenic human THP-1 cells [a monocytic-like leukemia cell line; #TIB-202, American Type Culture Collection (ATCC)] and THP1-XBlue-defMyD cells (#thpx-dmyd, InvivoGen) were cultured in RPMI 1640 (#FG1215, Biochrom) supplemented with 10% fetal bovine serum (#S0615, Biochrom). THP-1 cells were seeded from frozen ATCC stocks (5 × 10 6 cells/ml). Cells were incubated at 37°C in an atmosphere of 5% CO 2 . Subculturing was done by adding fresh medium to reach a cell concentration of 2 × 10 5 cells/ml. Medium was completely renewed every week. Human primary macrophages were isolated from at least four individual buffy coats donated by healthy volunteers (provided by DRK Berlin with informed consent for research). Peripheral blood monocytes were isolated from buffy coats with Ficoll-Paque and MACS Monocyte Isolation Kit II with MACS LS columns (Miltenyi Biotec). Primary monocytes were cultured and differentiated to macrophages in RPMI 1640 (#FG1215, Biochrom) supplemented with 10% human serum (Human Serum Type AB, P30-2501, PAN-Biotech).

Fischer C., Metsger M., Bauch S., Vidal R., Böttcher M., Grote P., Kliem M, & Sauer S. (2019). Signals trigger state-specific transcriptional programs to support diversity and homeostasis in immune cells. Science signaling, 12(581).

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Cell Line Maintenance and Viability

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The human tumor cell lines used were A375-C5 (melanoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). A374-C5, MCF-7, and NCI-H460 were grown in RPMI-1640 (Biochrom, Berlin, Germany), and U251, U373, and U87MG in DMEM (Biochrom). Media were supplemented with 5% heat-inactivated fetal bovine serum (FBS, Biochrom). Cell lines were grown at 37 °C in a 5% CO₂ humidified atmosphere (Hera Cell, Heraeus), and cell viability was monitored by Trypan Blue exclusion assay to ensure at least 95% viability before use.

Alves A., Correia-da-Silva M., Nunes C., Campos J., Sousa E., Silva P.M., Bousbaa H., Rodrigues F., Ferreira D., Costa P.C, & Pinto M. (2019). Discovery of a New Xanthone against Glioma: Synthesis and Development of (Pro)liposome Formulations. Molecules, 24(3), 409.

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Differentiation of THP-1 Macrophages

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The monocytic cell line THP-1 (DSMZ No. ACC 16) was cultured in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented with 10% FBS superior (Biochrom AG), 2 mM l-glutamine (Biochrom AG), 1 mM Na-pyruvate (Biochrom AG), gentamycin (10 µg/mL) (Biochrom AG), and 1 mM HEPES buffer (Biochrom AG) at 37 °C in a 5% CO₂ humidified atmosphere and passaged 2–3 times per week. Cells were used up to passage 20. To perform infection experiments, cells were differentiated into macrophages by stimulation with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Munich, Germany) 48 h prior to infection. For that purpose, cells were seeded in a 10 µM PMA solution in RPMI including listed supplements but without antibiotics at a density of 1 × 10⁶ cells per well, applying a 1.5-mL volume together with a six-well cell culture plate (Sarstedt AG, Nümbrecht, Germany). After 24 h of PMA stimulation, the stimulus was removed by washing the adherent cell layer with PBS. Medium including listed supplements but without antibiotics was provided for another 24 h before using THP-1-derived macrophages for infection experiments.

Sharbati S., Ravon F., Einspanier R, & zur Bruegge J. (2019). Mycobacterium smegmatis But Not Mycobacterium avium subsp. hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β. Microorganisms, 7(3), 63.

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RNA-Seq of Jurkat T cells under centrifugal stress

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Samples were generated, processed and sequenced as described previously (Vahlensieck et al., 2021a (link)). Briefly, Jurkat T cells (ATCC Manassas, United States, Clone E6-1, TIB152™) were cultured in RPMI 1640 (Biochrom, Berlin, Germany Cat. Nr FG1215) medium, supplemented with 10% FCS and 1% Pen/Strep, without centrifugation steps. The concentration was adjusted to 5 × 10⁶ cells/mL, samples were transferred into sterile, prewarmed (36.5°C) 2 ml pipettes and centrifuged using a custom-built 9xg pipette centrifuge provided by KEK (Bad Schmiedeberg, Germany). 10 samples per condition were generated. Pipettes were drained into 5 ml sterile, RNAse/DNase-free plastic tubes. RNA was extracted, poly(A) enriched with the help of the NEBNext Poly(A) mRNA Magnetic Isolation Module, library prepped with the NEBNext Ultra II RNA Library Prep Kit, both from New England Biology (Ipswich, United States), and sequenced (RNA-Seq) with appropriate quality control and standardization. There were three conditions, one control condition with 15 min at 1xg, one with 15 min at 9xg and one with 12 min at 1xg plus 3 min at 9xg (see results part for the underlying rationale).

Vahlensieck C., Thiel C.S., Pöschl D., Bradley T., Krammer S., Lauber B., Polzer J, & Ullrich O. (2022). Post-Transcriptional Dynamics is Involved in Rapid Adaptation to Hypergravity in Jurkat T Cells. Frontiers in Cell and Developmental Biology, 10, 933984.

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Reactivation and Mass Cytometry of Cryopreserved PBMCs

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Short-term reactivation of cryopreserved PBMCs and subsequent mass cytometry analysis were performed as described recently (19 (link)). In short, PBMCs were kept in liquid nitrogen before thawing in a 37 °C water bath. Cells were resuspended in cell culture medium (CCM: RPMI-1640, 10% fetal calf serum (FCS; Biochrom), 1x L-glutamine, 1x penicillin/streptomycin (both life technologies)) supplemented with 1:10'000 benzonase (Sigma), and centrifuged (300 rcf, 7 min, 24 °C). Samples were then rested overnight at 37 °C before restimulation with 50 ng mL-1 phorbol 12-myristate 13-acetate (PMA; Sigma) and 500 ng mL-1 ionomycin (Sigma) in the presence of 1x BrefeldinA (BD) for 4 hours at 37 °C.

Galli E., Hartmann F.J., Schreiner B., Ingelfinger F., Arvaniti E., Diebold M., Mrdjen D., van der Meer F., Krieg C., Al Nimer F., Sanderson N., Stadelmann C., Khademi M., Piehl F., Claassen M., Derfuss T., Olsson T, & Becher B. (2019). GM-CSF and CXCR4 Define a T Helper Cell Signature in Multiple Sclerosis. Nature medicine, 25(8), 1290-1300.

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PBMC Isolation and Culture

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PBMC were isolated by using density centrifugation as described elsewhere [28 (link)]. In brief, PBMC consisting mainly of lymphocytes and a small percentage of monocytes were separated from whole-blood in a 30 min centrifugation step at 1500 rpm at 4 °C by using Biocoll separation solution (MedPro, Vienna, Austria). After separation, PBMC were washed twice in phosphate-buffered saline (PBS, Serva, Heidelberg, Germany) solution containing 1 mM ethylenediaminetetraacetate (EDTA) (Merck, Vienna, Austria) and cultivated at a density of 1.0 × 10⁶ cells/ml in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% of heat-inactived fetal bovine serum (FBS, Invitrogen, Vienna, Austria), l-glutamine (final concentration: 2 mM) and gentamicin (final concentration: 50 µg/ml; both Serva, Heidelberg, Germany). Cells were incubated in a humidified atmosphere (37 °C and 5% CO₂) for 48 h.

Gostner J.M., Raggl E., Becker K., Überalla F., Schennach H., Pease J.E, & Fuchs D. (2015). Bisphenol A suppresses Th1-type immune response in human peripheral blood mononuclear cells in vitro. Immunology letters, 168(2), 285-292.

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Cultivating Cell Lines for Research

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A2780, IGROV1, OVCAR3, OVCAR4, OVCAR5, OVCAR8 and SKOV3 cells were purchased from the NCI-60 cell panel. 2102EP cell line was kindly provided by T. Mueller, Department of Oncology, University of Halle, Germany. NTERA-2D1 cell line was obtained from LGS Standards (Wesel, Germany). A549, H460, Calu-1, H23 and TOV-112D cells were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultivated in RPMI-1640 (Biochrom, Berlin, Germany) supplemented with 10% FCS and glutamine.
Peripheral blood mononuclear cells (PBMNCs) and fibroblasts were isolated and cultivated as described.^{51 (link), 52 (link)} The local ethics committee approved the investigation (project number 159/2011BO2) and informed consent was obtained from the patients.

Weilbacher A., Gutekunst M., Oren M., Aulitzky W.E, & van der Kuip H. (2014). RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38. Cell Death & Disease, 5(7), e1318-.

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