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Roswell park memorial institute (rpmi)

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RPMI is a powdered cell culture medium designed for the in vitro cultivation of a variety of cell types, including human cells. It provides a balanced salt solution and essential nutrients required for cell growth and maintenance.

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Lab products found in correlation

Antibacterial antimycotic, by Thermo Fisher Scientific (2 mentions) Ack lyses buffer, by Quality Biological (2 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Omega Scientific (2 mentions) Roswell park memorial institute (rpmi), by Omega Scientific (2 mentions) M csf, by R&D Systems (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Quality Biological (1 mentions) L glutamine, by Quality Biological (1 mentions) Flow cytometry permeabilization buffer, by R&D Systems (1 mentions) Percp labeled cd45, by BD (1 mentions) Facscan, by BD (1 mentions) Collagenase a, by Roche (1 mentions) Ack buffer, by Quality Biological (1 mentions)

4 protocols using roswell park memorial institute (rpmi)

1

Macrophage Differentiation and Adipose Interaction

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All steps were performed using sterile techniques. Femurs were collected in RPMI (Life Technologies, Inc.). Using a needle and syringe, marrow was flushed into RPMI containing 10% FBS (Omega Scientific, Inc.) and 5% antibacterial/antimycotics (Life Technologies, Inc.). Red blood cells were lysed using ACK lyses buffer (Quality Biological) and lysis was neutralized with complete RPMI. Bone marrow cells were differentiated into macrophages using MCSF (10ng/ml; R&D) and L929 conditioned media. Non-adherent cells were collected on day 7, counted and replated. BMDMs were treated on day 8. Cells were primed by four hour treatment with ultrapure LPS (1ug/ml;Sigma) alone; inflammasome stimulation was provided by treatment with ATP (5mM; 1hr). For co-culture with adipose, BMDMs were treated as described, media removed, cells washed and weighed VAT added to culture. VAT was stimulated with 1uM NE after 1 hour, and glycerol or FFA was measured at assay end.
Camell C.D., Sander J., Spadaro O., Lee A., Nguyen K.Y., Wing A., Goldberg E.L., Youm Y.H., Brown C.W., Elsworth J., Rodeheffer M.S., Schultze J.L, & Dixit V.D. (2017). Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis in the aged. Nature, 550(7674), 119-123.
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2

Macrophage Differentiation and Adipose Interaction

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All steps were performed using sterile techniques. Femurs were collected in RPMI (Life Technologies, Inc.). Using a needle and syringe, marrow was flushed into RPMI containing 10% FBS (Omega Scientific, Inc.) and 5% antibacterial/antimycotics (Life Technologies, Inc.). Red blood cells were lysed using ACK lyses buffer (Quality Biological) and lysis was neutralized with complete RPMI. Bone marrow cells were differentiated into macrophages using MCSF (10ng/ml; R&D) and L929 conditioned media. Non-adherent cells were collected on day 7, counted and replated. BMDMs were treated on day 8. Cells were primed by four hour treatment with ultrapure LPS (1ug/ml;Sigma) alone; inflammasome stimulation was provided by treatment with ATP (5mM; 1hr). For co-culture with adipose, BMDMs were treated as described, media removed, cells washed and weighed VAT added to culture. VAT was stimulated with 1uM NE after 1 hour, and glycerol or FFA was measured at assay end.
Camell C.D., Sander J., Spadaro O., Lee A., Nguyen K.Y., Wing A., Goldberg E.L., Youm Y.H., Brown C.W., Elsworth J., Rodeheffer M.S., Schultze J.L, & Dixit V.D. (2017). Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis in the aged. Nature, 550(7674), 119-123.
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3

Cell Line Characterization and Culture

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Cell lines were tested for mycoplasma and identities were ensured by RNA-seq and genotyping. RMS cell lines RH4 and RH30 were gifts from Dr. Peter Houghton, SCMC from Dr. Janet Shipley, RD, and CTR from Dr. Lee Helman. Osteosarcoma cell lines OSA and HU09 and Ewing’s Sarcoma cell lines TC-32 and A673 were from Dr. Paul Meltzer. Human fibroblast cell line 7250 (CRL-7250) was obtained from ATCC. All cell lines except OSA and HU09 were grown in DMEM (Quality Biological, #112-013-101CS) supplemented with 10% FBS (ThermoFisher, #A31605-02), 2 mM L-glutamine (Quality Biological, #118-288-061), and 100 U/ml penicillin/streptomycin (ThermoFisher, #15140122). OSA and HU09 were grown in RPMI (Quality Biological, #112-024-101CS) with the same supplements as DEME.
Kim Y.Y., Gryder B.E., Sinniah R., Peach M.L., Shern J.F., Abdelmaksoud A., Pomella S., Woldemichael G.M., Stanton B.Z., Milewski D., Barchi JJ J.r., Schneekloth JS J.r., Chari R., Kowalczyk J.T., Shenoy S.R., Evans J.R., Song Y.K., Wang C., Wen X., Chou H.C., Gangalapudi V., Esposito D., Jones J., Procter L., O’Neill M., Jenkins L.M., Tarasova N.I., Wei J.S., McMahon J.B., O’Keefe B.R., Hawley R.G, & Khan J. (2024). KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma. Nature Communications, 15, 1703.
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4

Lung Single Cell Analysis by FACS

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Whole lung single cell suspension was analyzed by FACS analysis with. Briefly mouse lungs were harvested then digested in lung digestion media with 1.0 mg/mL collagenase A (Roche Diagnostics) in RPMI (Quality Biological), and erythrocytes were lysed with ACK buffer (Quality Biological). Cells isolated from the lungs were stained with PerCP-labeled CD45 (BD), APC-Cy7-labeled CD11b (BD), PE-labeled Ly6G (BD), and FITC-Labeled LTA4H (Bioss). Infiltrating leukocytes were gated from non-leukocytes by the expression of CD45. Next, all CD45high cells were gated into Ly6Ghigh and CD11bhigh cells (neutrophils) as previously described.(3 (link)) For the purpose of detecting cells containing the LTA4H in their intra-cellular space, cells were permeabilized with flow cytometry permeabilization buffer (R&D) after stained with CD45 antibody. Cells expressing LTA4H were separated into two groups, leukocytes (CD45high) and non-leukocytes (CD45low). Multi-color detection of the stained cells was performed on a FACScan flow cytometer (BD Biosciences). FlowJo (version 8.8.6.) was used to analyze the data.
Paige M., Wang K., Burdick M., Park S., Cha J., Jeffrey E., Sherman N, & Shim Y.M. (2014). Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5059-5068.
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