Complete rpmi 1640

Manufactured by Lonza

Sourced in United States, Switzerland, Spain

Complete RPMI 1640 is a cell culture medium that provides a complete and balanced formulation of nutrients, vitamins, amino acids, and other essential components required for the growth and maintenance of a variety of cell types in vitro.

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Lab products found in correlation

Fetal bovine serum (fbs), by Merck Group (2 mentions) Phytohemagglutinin (pha), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Il 15, by Immunotools (1 mentions) Interleukin 7 (il 7), by Immunotools (1 mentions) L glutamax, by Merck Group (1 mentions) Human ab serum, by Merck Group (1 mentions) Ficoll hypaque, by GE Healthcare (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Lps from e coli o111 b4, by Merck Group (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) α il 4, by Thermo Fisher Scientific (1 mentions) Ifn γ antibody, by Thermo Fisher Scientific (1 mentions) Easysep isolation kits, by STEMCELL (1 mentions)

9 protocols using complete rpmi 1640

Generation and Maintenance of Human iNKT Clones

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Peripheral blood mononuclear cells (PBMC) were isolated from fresh human blood by density gradient centrifugation (Ficoll-Hypaque; GE Healthcare).
Human iNKT clones were generated from PBMCs as previously described (10 (link)). Briefly, single live CD3⁺Vα24⁺Vβ11⁺ T cells were sorted into 96-well plates using FACSAria and cultured in the presence of 1ug/ml phytohaemagglutinin (PHA) (Sigma) and γ-irradiated (35Gy) autologous feeder PBMCs in complete RPMI 1640 (Lonza) containing 10% FBS (Sigma), 200IU IL-2 (Proleukin, Chiron), 50ng/ml IL-7, 50ng/ml IL-15 (Immunotools), 1mM sodium pyruvate, 1% non-essential amino acids, 1% L-glutamax, 50uM 2-mercaptoethanol, 100IU penicillin, 100ug/ml streptomycin and 2% human AB serum (all Sigma).
T2 lymphoblasts (T2-) and stably CD1d-expressing T2 lymphoblasts (T2-CD1d) were maintained in complete medium (RPMI (10% FBS, 1% non-essential amino acids, 1% L-glutamax, 1mM sodium pyruvate, 100IU penicillin and 100ug/ml streptomycin).

Mansour S., Tocheva A.S., Sanderson J.P., Goulston L.M., Platten H., Serhal L., Parsons C., Edwards M.H., Woelk C.H., Elkington P.T., Elliott T., Cooper C., Edwards C.J, & Gadola S.D. (2015). Structural and functional changes of the iNKT clonal repertoire in early Rheumatoid Arthritis. Journal of immunology (Baltimore, Md. : 1950), 195(12), 5582-5591.

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Stimulation of PBMCs with LPS, Pam3Cys, and ATP

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PBMCs were isolated from fresh EDTA-anticoagulated blood by density gradient centrifugation in Ficoll-Paque PLUS (GE Healthcare, UK). After a minimum of 3 h rest in complete RPMI 1640 (Lonza, Basel, Switzerland) cells were stimulated with 1 μg/ml lipopolysaccharides (LPS) from E.coli O111:B4 (Sigma), 1 μg/ml Pam3Cys-SKKKK (Pam₃Cys) (EMC microcollections), and 5 mM ATP (stock solution neutralized; Sigma) for the indicated times.

Keskitalo S., Haapaniemi E., Einarsdottir E., Rajamäki K., Heikkilä H., Ilander M., Pöyhönen M., Morgunova E., Hokynar K., Lagström S., Kivirikko S., Mustjoki S., Eklund K., Saarela J., Kere J., Seppänen M.R., Ranki A., Hannula-Jouppi K, & Varjosalo M. (2019). Novel TMEM173 Mutation and the Role of Disease Modifying Alleles. Frontiers in Immunology, 10, 2770.

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Murine T Cell Activation Assay

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Purified α-CD3 (145-2C11), purified α-CD28, α-CD25 (3C7), Biotin α-CD25, fluorescein isothiocyanate (FITC)-α-CD4, phycoerythrin (PE)-α-CD25, unconjugated, (allophycocyanin) APC, Alexafluor-647 or PE conjugated α-CD44, APC, Alexafluor 647, PE or PE-Cy7 conjugated IL-17-A, Foxp3 antibodies, IL-6 blocking antibodies, α-IL-4 and IFN-γ antibodies, were all purchased from eBiosciences (San Diego, CA). Mouse CD4 isolation kit II was purchased from Miltenyi Biotec (Auburn, CA). EasySep isolation kits were purchased from Stem Cell technologies (Canada). Human IL-2 purchased from NCI, was a kind gift from Mike Lenardo lab (NIAID, NIH). Recombinant mouse IL-6 was purchased from BioBasic Inc (Amherst, NY), and human TGF-β was purchased from R&D Systems (Minneapolis, MN). Mouse cells were cultured in complete RPMI-1640 (Bio-Whittaker) supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 mM Glutamax (Invitrogen) and 50 μM β-mercaptoethanol.

Bhaskaran N., Cohen S., Zhang Y., Weinberg A, & Pandiyan P. (2015). TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis. Pathogens, 4(1), 90-110.

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Hypoxia-Induced Cell Migration Assay

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786-O cells with the same number of passages (at least 20 passages) grown in normoxic and hypoxic conditions were used for assay. 4 × 10⁴ cells were suspended in 300 μL serum-free RPMI 1640 and poured into 24-well hanging inserts (0.8 μm, EMD Millipore Corporation, Billerica, MA, USA) and 500 μL complete RPMI 1640 (Lonza) was added to the lower wells. After 22 h, the insert membranes were stained with 0.2% crystal violet in 20% methanol. The cells on the inner side were wiped off with cotton swabs and the migrated cells on the outside membrane were counted. The results were derived from at least three independent experiments.

Chen S.C., Chen F.W., Hsu Y.L, & Kuo P.L. (2017). Systematic Analysis of Transcriptomic Profile of Renal Cell Carcinoma under Long-Term Hypoxia Using Next-Generation Sequencing and Bioinformatics. International Journal of Molecular Sciences, 18(12), 2657.

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Isolation and Culture of Splenic Macrophages

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Mouse spleens were physically disrupted in 10 ml complete RPMI-1640 (Lonza, Walkersville, MD), supplemented with 2 mM L-glutamine, 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 50 IU/ml penicillin and 50 µg/ml streptomycin (MP Biomedicals, Irvine, CA). The splenocyte suspension was passed through a 40 µm nylon cell strainer (Fisher Scientific, Fair Lawn, NJ) and cultured in a 100 × 15 mm petri dish (Fisher) in a 5% CO_2, 37ºC humidified incubator (Forma Scientific, Marietta, OH). After 3 days, the non-adherent cells were removed. On day 6, the culture supernatant was discarded and the adherent cells (largely splenic macrophages) were washed three times with Dulbecco’s Ca²⁺/Mg²⁺-free phosphate-buffered saline (DPBS; HyClone, Logan, UT). After the last wash, 3 ml of Cellstripper non-enzymatic cell dissociation solution (Mediatech, Manassas, VA) was added and cells were incubated at 5% CO₂, 37ºC for 10 min on an orbital shaker. Macrophages were lifted by gentle pipetting and added to 10 ml ice-cold complete RPMI-1640. Cells were centrifuged at 350 g for 10 min and resuspended in media at the desired density. All the described experiments were performed using splenic macrophages differentiated for 6 days except for patch clamp experiments (see below).
Peritoneal macrophages were isolated as previously described in detail [12 (link)].

Beesetty P., Rockwood J., Kaitsuka T., Zhelay T., Hourani S., Matsushita M, & Kozak J.A. (2021). Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice. The FEBS journal, 288(11), 3585-3601.

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Murine Splenocyte Cytokine Profiling

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Spleens from experimental mice were collected and processed into a single cell suspension. 0.7 × 10⁶ splenocytes were plated in triplicate in 96-well plates in the presence of 10 μg/mL rhIDUA in complete RPMI 1640 (Lonza, Switzerland) with 10% fetal bovine serum (FBS) (Euroclone, ECS0180L), 100 U/ml penicillin/streptomycin (Lonza, 17-602E), 2 mM L-glutamine (Lonza, 17-605E), Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA) (Gibco, 11140-035), 1 mM Sodium Pyruvate (Gibco, 11360-039), 50 nM 2-Mercaptoethanol (Gibco, 31350-010). After 96 h of culture, supernatants were collected and tested for cytokine production with Bio-Plex Pro Mouse Cytokine Th1/Th2 assay (Bio-Rad), according to the manufacturer’s instructions.

Squeri G., Passerini L., Ferro F., Laudisa C., Tomasoni D., Deodato F., Donati M.A., Gasperini S., Aiuti A., Bernardo M.E., Gentner B., Naldini L., Annoni A., Biffi A, & Gregori S. (2019). Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease. Molecular Therapy, 27(7), 1215-1227.

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Cell Culture Protocols for T and B Cells

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All T and B cell lines were cultured in complete RPMI 1640 (Lonza) supplemented with 10–20% heat-inactivated fetal calf serum (Valley Biomed, HyClone), 2 mM glutamine, and 100U/ml each of penicillin and streptomycin (Life Technologies). Jurkat T cells (E6.1) and BJAB B cell lines were purchased from the American Tissue Type Collection. JPM50.6 T cells and MALT1-knockdown (K_D) Jurkat T cells were kindly provided by Dr. Lawrence Kane (University of Pittsburgh). NEMO-deficient JM.4.5.2 T cells were a gift from Dr. Brian Schaefer (USUHS). DLBCL lines (OCI-Ly3, WSU-NHL, U2932, HBL-1) were kindly provided by Dr. Louis Staudt (NCI, NIH). In some experiments, T cell lines were stimulated using 1 μg/ml each of anti-CD3e and anti-CD28 agonistic Abs (BD Biosciences).

Stinson J.R., Dorjbal B., McDaniel D.P., David L., Wu H, & Snow A.L. (2020). Gain-of-function mutations in CARD11 promote enhanced aggregation and idiosyncratic signalosome assembly. Cellular immunology, 353, 104129.

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Murine Cancer Cell Line Authentication

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The CT26 (BALB/c) murine colon carcinoma cell lines were purchased from and authenticated by the ATCC. MC38 (C57/Bl6) cells were obtained from and authenticated by the NIH. MCA-205 cells were kindly provided by Dr. Andrew Weinberg (Earle A. Chiles Research Institute). CT26 cells were maintained in complete RPMI 1640 (Thermo Fisher Scientific), MC38 cells in DMEM (Thermo Fisher Scientific), and MCA-205 cells in complete RPMI 1640 [10% FBS, 10 mmol/L HEPES, 1% nonessential amino acids, 1% sodium pyruvate (Lonza), and penicillin (100 IU/mL)-streptomycin (100 mg/mL)-glutamine (29.2 mg/mL; Invitrogen)]; the identity of this cell line was verified through monthly assessment of morphology and growth kinetics; in vitro MC38 cells were supplemented with 1% nonessential amino acids and 1% sodium pyruvate (Millipore Sigma). Hs5 cells were obtained from and authenticated by the ATCC, and were grown in SFEM2 media (Stem Cell Technologies) for use in generation of bone marrow-derived macrophages (BMDM). L-929 cells were obtained from and authenticated by the ATCC, and were grown in DMEM þ 10% FBS. All cell lines were used at low passage (<10 passages in vitro after receipt from source). All cell lines were tested and screened negative for Mycoplasma using the MycoAlert test (Lonza).

Badeaux M.D., Rolig A.S., Agnello G., Enzler D., Kasiewicz M.J., Priddy L., Wiggins J.F., Muir A., Sullivan M.R., Van Cleef J., Daige C., Vander Heiden M.G., Rajamanickam V., Wooldridge J.E., Redmond W.L, & Rowlinson S.W. (2021). Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth. Cancer immunology research, 9(4).

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Peripheral Blood Mononuclear Cell Isolation and Stimulation

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PBMCs were isolated from blood collected in heparinized tubes by density gradient centrifugation using Histopaque ® 1.077 (Sigma, Madrid, Spain). PBMCs were washed and suspended in complete RPMI-1640 (Lonza, Barcelona, Spain) (cRPMI) plus 10% foetal bovine serum (FBS) (Sigma, Madrid, Spain); cell viability was assessed with Trypan blue staining. Then, PBMCs were seeded into 96-well plates (1 x 10 6 cells/well) and incubated with baculovirus-expressed PCV2 Cap protein (final concentration per well: 0.6 µg/mL); phytohemagglutinin (Sigma, Madrid, Spain) (final concentration per well: 10 µg/mL) as a positive control; or cRPMI plus 10% FBS as a negative control for 24 h at 37°C in a 5% humidified CO2 atmosphere. After incubation, plates were centrifuged and cell culture supernatants were collected and stored at -80ºC until further examination.

Oliver-Ferrando S., Segalés J., Sibila M, & Díaz I. (2018). Comparison of cytokine profiles in peripheral blood mononuclear cells between piglets born from Porcine circovirus 2 vaccinated and non-vaccinated sows. Veterinary microbiology, 214.

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