Rpmi 1640 medium

Manufactured by Biochrome

Sourced in Germany, United Kingdom

RPMI 1640 medium is a commonly used cell culture medium formulation. It provides a balanced salt solution and essential nutrients to support the growth and maintenance of a variety of mammalian cell types in vitro.

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Lab products found in correlation

β mercaptoethanol, by Merck Group (4 mentions) Hepes, by MP Biomedicals (3 mentions) Non essential amino acids (neaa), by Merck Group (3 mentions) Sodium pyruvate, by Merck Group (3 mentions) Penicillin, by Merck Group (2 mentions) Streptomycin, by Merck Group (2 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) Fetal calf serum (fcs), by Biochrome (1 mentions) Edta coated tube, by Sarstedt (1 mentions) 48 well plate, by Thermo Fisher Scientific (1 mentions) Luna cell counter, by Biozym (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Trypsin edta, by Merck Group (1 mentions) Bxpc 3, by American Type Culture Collection (1 mentions) Aspc 1, by American Type Culture Collection (1 mentions) Mia paca 2, by American Type Culture Collection (1 mentions) Fetal bovine serum (fbs), by Biochrome (1 mentions) Anti cd28, by Thermo Fisher Scientific (1 mentions) Easysep mouse t cell isolation kit, by STEMCELL (1 mentions) α cd3 145 2c11, by BioXCell (1 mentions)

Close spelling variants of this product

RPMI-1640 (5 citations)

12 protocols using rpmi 1640 medium

THP-1 Cells for In Vitro Mycobacterial Infection

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THP-1 cells were used for the in vitro infection and intervention experiments. These cells were previously shown to be a suitable alternate of hMDMsfor the in vitro infection experiments with mycobacteria (Bosshart and Heinzelmann, 2016 (link); Madhvi et al., 2019 (link)). We preferred to use THP-1 cells since they are easier to revive and more stable for in vitro experiments. Commercially available THP-1 cells (ATCC-88081201) [European Collection of Authenticated Cell Cultures (ECACC), Salisbury, UK] were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (Biochrome, Germany). THP-1 cells were treated with a final concentration of 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich, USA) for 48 h. For infection experiments, THP-1 cells were seeded in 48-well plates (Greiner Bio-One, cat. No. 677180) with 0.07 x 10 (John et al., 2018 (link)) cells per well.

Madhvi A., Mishra H., Chegou N.N, & Baker B. (2022). Increased Interferon-Induced Protein With Tetracopeptides (IFITs) Reduces Mycobacterial Growth. Frontiers in Cellular and Infection Microbiology, 12, 828439.

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Evaluation of Inflammatory Response in Murine Lung

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Anesthetized mice were euthanized by exsanguination at days 1, 3 and 7 after CNP instillation. Blood was withdrawn from the retro orbital plexus with a capillary and collected in EDTA coated tubes (Sarstedt, Germany) for hematological analysis (ADVIA Hematology Systems (Bayer Diagnostics). Lungs were lavaged with 10 × 1 mL phosphate buffered saline (PBS, calcium and magnesium free) as described previously [38 (link)]. The BAL fluids from the first two lavages were used for protein analysis after cell separation by centrifugation (425 × g, 20 min at room temperature). The cell pellet was resuspended in 1 mL RPMI 1640 medium (BioChrome, Berlin, Germany) supplemented with 10% fetal calf serum (Seromed, Berlin, Germany). Cell viability was determined by the Trypan blue dye exclusion test. Differential cell counts were performed on cytospin preparations (May-Grünwald-Giemsa staining; 2 × 200 cells were counted).

Geiser M., Stoeger T., Casaulta M., Chen S., Semmler-Behnke M., Bolle I., Takenaka S., Kreyling W.G, & Schulz H. (2014). Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis. Particle and Fibre Toxicology, 11, 19.

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Lung Tissue Culture for Nodule Research

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HLT was taken after informed consent from patients (mean age 63 years; 3 males, 1 female) without underlying chronic or acute lung diseases undergoing lung resections because of pulmonary nodules as previously described [22] . The study was approved by the Ethics Committee of the University of Lübeck and is in compliance with the Declaration of Helsinki. Tumour-free pieces were taken at least 5 cm away from the tumour front. Pieces of 0.3-0.4 g were cultured for 24 h in RPMI 1640 medium (Biochrome AG) containing 10% FCS in 48-well plates (Nunc) at 37 ° C and 5% CO 2 .

Wagner C., Goldmann T., Rohmann K., Rupp J., Marwitz S., Rotta Detto Loria J., Limmer S., Zabel P., Dalhoff K, & Drömann D. (2015). Budesonide Inhibits Intracellular Infection with Non-Typeable Haemophilus influenzae Despite Its Anti-Inflammatory Effects in Respiratory Cells and Human Lung Tissue: A Role for p38 MAP Kinase. Respiration; international review of thoracic diseases, 90(5).

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Cell Line Cultivation and Quantification

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Permanent cell lines BxPC3, MIA PaCa-2 and AsPC-1 were obtained from the American Type Culture Collection (Rockville, MD, USA). The BH1362 KRAS G12C NSCLC cell line was established from a pleural effusion according to the Ethics Committee EK-21-210-1221 of the Viennese Hospital Association. Aforesaid cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS (Biochrome, Berlin, Germany) and Penicillin/Streptomycin (Sigma-Aldrich). Upon confluence cells were detached with trypsin/EDTA (Sigma-Aldrich) and counted with a LUNA cell counter (Biozym, Vienna, Austria).

Plangger A., Rath B., Stickler S., Hochmair M., Lang C., Weigl L., Funovics M, & Hamilton G. (2022). Cytotoxicity of combinations of the pan-KRAS SOS1 inhibitor BAY-293 against pancreatic cancer cell lines. Discover. Oncology, 13, 84.

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T Cell Isolation and Proliferation Assay

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Whole spleen cells were obtained from C57BL/6 mice and T cells were isolated from whole spleen cells using the EasySep TM Mouse T Cell Isolation Kit (Stemcell, Vancouver, BC, Canada, 19851) according to the manufacturer's protocol. T cells were cultured in RPMI 1640 medium (Biochrome, Berlin, Germany) supplemented with 10% fetal calf serum (Linaris, Dossenheim, Germany), 10 mmol/L HEPES (MP Biomedicals, California, USA), 1 mmol/L sodium pyruvate (Sigma), 1× non-essential amino acids (Sigma, Darmstadt, Germany), and 10 mmol/L β-mercaptoethanol (Sigma). Splenocytes were plated at a density of 0.5 × 10 6 per well (96-well plate) and stimulated with interleukin-2 (IL-2) (1 ng/mL; Novoprotein, CK24), anti-CD28 (1 μg/mL; eBioscience, San Diego, USA, 102101), and anti-mouse CD3ε antibody (7.5 μg/mL, eBioscience). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to assess the in vitro proliferation of stimulated T cells.

Yuan W., Zhang H., Peng L., Chen C., Feng C., Tang Z., Cui P., Li Y., Li T., Qiu X., Cui Y., Zeng Y., Luo J., Xie X., Guo Y., Jiang X, & Dai H. (2024). Inhibition of interferon regulatory factor 4 orchestrates T cell dysfunction, extending mouse cardiac allograft survival. Chinese medical journal.

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Murine Splenocyte Activation Kinetics

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C57BL/6 splenocytes were isolated and cultured in RPMI 1640 medium (Biochrome, Berlin, Germany) supplemented with 10% FCS (Linaris, Dossenheim, Germany), PenStrep (100U Penicillin, 100μg Streptomycin/ml; Sigma), 10mM HEPES (MP Biomedicals), 1mM sodium pyruvate (Sigma), 1x non-essential amino acids (Sigma) and 10μM β-mercaptoethanol (Sigma). To assess CD40L and CD69 expression kinetics, 0.5x10⁶ splenocytes per well (96 well plate) were either stimulated with phorbol myristate acetate (PMA) (10ng/ml)/Ionomycin (0.2µg/ml), with 10μg/ml αCD3 (145-2C11) (BioXcell) and 1μg/ml αCD28 (37.51) (BioLegend) or with 0.5x10⁶ irradiated allogeneic donor cells (Balb/c) (for the mixed lymphocyte reaction, MLR). Early T-cell activation was assessed via CD69 surface expression. CFSE and Ki-67 were used to follow in-vitro proliferation of stimulated T-cells. To capture “cycling” CD40L, a surface mobilization assay was performed as described in (28 (link)). Briefly, the fluorescence-labeled anti-CD40L antibody was added directly into the medium during the last 4 hours of cell culture. In this way, both intracellular CD40L cycling to the cell surface during the incubation time and cell surface-expressed of CD40L were detected (total CD40L expression).

Unger L.W., Muckenhuber M., Mahr B., Schwarz C., Pilat N., Granofszky N., Regele H, & Wekerle T. (2022). Chronic CD40L blockade is required for long-term cardiac allograft survival with a clinically relevant CTLA4-Ig dosing regimen. Frontiers in Immunology, 13, 1060576.

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Phl p 5-specific Splenocyte Culture

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Duplicates of 10⁶ Phl p 5-coupled splenocytes were cultured in Greiner Cellstar 24-well plates (Sigma-Aldrich, Burlington, MA, USA) in RPMI 1640 medium (Biochrome, Cambridge, UK) supplemented with 10% FCS (Linaris, Frankfurt, Germany), PenStrep (100 U penicillin, 100 μg streptomycin/mL; Sigma), 10 mM HEPES (MP Biomedicals, Santa Ana, CA, USA), 1 mM sodium pyruvate (Sigma-Aldrich), 1x non-essential amino acids (Sigma-Aldrich), and 10 μM β-mercaptoethanol (Sigma-Aldrich). Cultures were incubated for 48 h at 37 °C and 5% CO_2. The harvested cells were prepared for flow and image cytometry.

Mengrelis K., Niederacher G., Prickler L., Kainz V., Weijler A.M., Rudolph E., Stanek V., Eckl-Dorna J., Baranyi U., Spittler A., Focke-Tejkl M., Bohle B., Valenta R., Becker C.F., Wekerle T, & Linhart B. (2024). Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy. Cells, 13(5), 446.

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Mast Cell Activation Assay

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RBL-2H3 cell subline was cultured as described previously [25 (link)] in RPMI 1640 medium (Biochrome AG, Berlin, Germany) containing 10% foetal calf serum. 6 × 10⁴ cells were plated in 96-well tissue culture plates (Greiner, Bio-One frickenhaus, Germany), loaded with 1 : 30 diluted mouse sera and incubated for 2 h at 37 °C and 5% CO₂. Supernatants were removed and the cell layer was washed 2× with Tyrode′s buffer (137 mm NaCl, 2.7 mm KCL, 0.5 mm MgCl₂, 1.8 mm CaCl₂, 0.4 mm NaH₂PO₄, 5.6 mm D-glucose, 12 mm NaHCO₃, 10 mm HEPES and 0.1% w/v BSA, pH 7.2). Preloaded cells were stimulated with rPhl p 5 or rBet v 1 (0.03 μg per well) for 30 min at 37 °C. The supernatants were analysed for ß-hexosaminidase activity by incubation with the substrate 80 μm 4-methylumbelliferyl-N-acetyl-ß-D-glucosamide (Sigma-Aldrich) in citrate buffer (0.1 m, pH 4.5) for 1 h at 37 °C. The reaction was stopped by addition of 100 μL glycine buffer (0.2 m glycine, 0.2 m NaCl, pH 10.7), and the fluorescence was measured at λ_ex: 360/λ_em: 465 nm using a fluorescence microplate reader (Wallac, Perkin Elmer, Vienna, Austria). Results are reported as percentage of total ß-hexosaminidase released after the addition of 1% Triton X-100. Determinations were performed in triplicates.

Gattringer M., Baranyi U., Pilat N., Hock K., Klaus C., Ramsey H.E., Wrba F., Valenta R, & Wekerle T. (2016). Anti-OX40L alone or in combination with anti-CD40L and CTLA4Ig does not inhibit the humoral and cellular response to a major grass pollen allergen. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 46(2), 354-364.

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Inflammatory Response in CNP-Induced Lung Injury

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3 h, 6 h, 12 h, 18 h, 1d, 3d and 7d, after CNP instillation, mice were anesthetized via intraperitoneal injection of a mixture of xylazine and ketamine and sacrificed by retrobulbar exsanguination. Immediately afterwards, BAL was performed by cannulating the trachea and infusing the lungs eight times with 1.0 ml PBS without calcium and magnesium, as described previously [12 , 71 ]. BAL fluids of the first two lavages were pooled for protein and cytokine analysis after BAL cell separation by centrifugation (425 g, 20 min at room temperature). The cell pellets were suspended in 1 ml RPMI 1640 medium (BioChrome, Berlin, Germany) supplemented with 10 % fetal calf serum (Seromed, Berlin, Germany), and the number of living cells was counted by the trypan blue exclusion method. BAL cell differentials were performed on cytospin preparations (May-Grünwald- Giemsa staining; 2 × 200 cells counted) and the number of polymorphonuclear neutrophils (PMNs) was used as a marker of inflammation [12 , 71 ].

Chen S., Yin R., Mutze K., Yu Y., Takenaka S., Königshoff M, & Stoeger T. (2016). No involvement of alveolar macrophages in the initiation of carbon nanoparticle induced acute lung inflammation in mice. Particle and Fibre Toxicology, 13, 33.

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Murine T-Cell Activation and Expansion

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Golgi Plug Protein Transport Inhibitor, Cytofix/Cytoperm Solution, PermWash buffer and 70 µm cell strainer were purchased from BD Pharmingen (Franklin Lakes, USA). Click.iT EdU flow cytometry assay kit and sytox AADvanced from Life Technologies Corporation (Carlsbad, USA). Murine MicroBeads CD4 (L3T4), CD8α (Ly2) and the CD4/CD8 T-cell activation/Expansion Kit mouse were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). LPS (L2880) were purchased from Sigma-Aldrich (St.Louis, USA). Vetbond tissue adhesive from 3 M (St. Paul, USA). EDTA-capillary and Li-Heparin-capillary tubes were acquired from Sarstedt (Nümbrecht, Germany). RPMI 1640 medium from Biochrome AG (Berlin, Germany). FCS from Biowest LLC (Kansas City, USA). 2-Mercapto-ethanol from Roth (Karlsruhe, Germany). VitaLyse Lysing Buffer from BioE (St. Paul, USA). Streptavidin from Dianova (Hamburg, Germany). 24-well plates from Corning Incorporated (Corning, USA). CFSE from Enzo Lifescience Inc. (Lörrach, Germany). Lauryl-maltoside (n-Dodecyl-ß-D-maltoside, ULTROL Grade) from Calbiochem (Bad Soden, Germany). CpG (TCCATGACGTTCCTGACGTT) was acquired from Sigma-Aldrich (St.Louis, USA). GP33–41 (KAVYNFATM) and GP61–80 (GLKGPDIYKGVYQFKSVEFD) peptides were synthesized by Bio-Synthesis (Louisville, USA).

Markwart R., Condotta S.A., Requardt R.P., Borken F., Schubert K., Weigel C., Bauer M., Griffith T.S., Förster M., Brunkhorst F.M., Badovinac V.P, & Rubio I. (2014). Immunosuppression after Sepsis: Systemic Inflammation and Sepsis Induce a Loss of Naïve T-Cells but No Enduring Cell-Autonomous Defects in T-Cell Function. PLoS ONE, 9(12), e115094.

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