Imdm glutamax 1

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IMDM Glutamax I is a basal medium designed for the cultivation of a wide range of cell types. It contains the amino acid L-glutamine, which is an essential nutrient for cell growth and metabolism.

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GlutaMAX (10236 citations) GlutaMAX-I (877 citations) IMDM + GlutaMAX (43 citations)

7 protocols using imdm glutamax 1

Culturing human cell lines

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K562 human immortalised chronic myelogenous leukaemia bone marrow cells were grown in IMDM + Glutamax-I (Gibco), supplemented with 10% fetal bovine serum (Sigma) and 1x Pen./Strep. (Sigma). Human cervix adenocarcinoma HeLa S3 cells were cultured in Ham’s F-12 nutrient mix with 2 mM l-Glutamine (Gibco), supplemented with 10% fetal bovine serum (Sigma) and 1x Pen./Strep. (Sigma). Human mammary epithelial MCF10a cells were cultured in DMEM/F-12 (Sigma) supplemented with 5% horse serum (Thermofisher), 20 ng/ml EGF (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma), 10 µg/ml insulin (Sigma), 2 mM l-Glutamine (Thermofisher) and 1x Pen./Strep. (Sigma)^{26 (link)}. All cells were grown at 37^oC, 5% CO2, and were regularly tested for mycoplasma presence.

Agirre E., Oldfield A.J., Bellora N., Segelle A, & Luco R.F. (2021). Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition. Nature Communications, 12, 682.

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Molting Assay for Anthelmintic Screening

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L3 stage larvae previously collected and cryopreserved in Cameroon were rapidly thawed in a 37°C water bath and washed in incomplete media comprised of a 1:1 ratio of Medium NCTC-109 and IMDM + GlutaMax-I containing 1X glutamine, penicillin, and streptomycin (all from Gibco by Life Technologies, Grand Island, NY). The number of worms was adjusted to about 10 worms per 50 μL in complete medium containing 20% heat inactivated FCS. Worms were distributed into the wells of a 96-well plate containing 50 μL of 1.5 × 10⁵ normal human PBMCs. 100 μL of 2X auranofin (final concentrations of 30 μM, 10 μM, 3 μM, 1 μM and 0.3 μM) were added to each well for a final volume of 200 μL. Each concentration was tested in triplicate. Controls included 0.05% DMSO in complete medium and complete medium only with neither DMSO nor compound added. The 96-well plates were then incubated at 37°C in a 5% CO₂ incubator for 6 days, then molting was assessed using an inverted microscope. Molting was determined in each well by counting the presence of fourth-stage larvae (L4) and empty casts of the L3. The percent inhibition of molting was calculated based on the number of treated larvae that were able to molt in comparison to the number of control larvae that had successfully molted. Prism 4.0 for Windows was used to calculate IC₅₀s.

Bulman C.A., Bidlow C.M., Lustigman S., Cho-Ngwa F., Williams D., Rascón, Jr A.A., Tricoche N., Samje M., Bell A., Suzuki B., Lim K.C., Supakorndej N., Supakorndej P., Wolfe A.R., Knudsen G.M., Chen S., Wilson C., Ang K.H., Arkin M., Gut J., Franklin C., Marcellino C., McKerrow J.H., Debnath A, & Sakanari J.A. (2015). Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis. PLoS Neglected Tropical Diseases, 9(2), e0003534.

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Sorting Subpopulations of WM Cell Line

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Cell Line and Sorting WM cell line, MWCL-1 was provided from Mayo Foundation for Medical Education and Research. MWCL-1 cells were cultured in IMDM+GlutaMAX-I (Gibco by Life Technologies, Carlsbad, USA) supplemented with 10% fetal bovine serum (Central America Origin, Biosera, Kansas City, USA). Cells were stained with CD20-Allophycocyanin (APC) (clone 2H7, BD Biosciences, San Jose, USA) and CD138-Phycoerythrin (PE) (clone MI15, BD Biosciences) antibodies, and CD20 - CD138 -, CD20 + CD138 -, CD20 + CD138 + subpopulations were sorted with FACS Aria II (BD Biosciences). We reacted cells in the presence of human FcR-blocking reagent (Miltenyi Biotec, Auburn, USA). The dot blot pattern obtained with FcR blocking was comparable to that without blocking. 23 To remove dead cells, propidium iodide (PI) staining and gating of forward-scatter-area (FSC-A) versus side-scatter-area (SSC-A) were performed. The proportion of dead cells removed by PI staining and gating of FSC-A versus SSC-A was almost same as that removed only by gating of FSC-A versus SSC-A. 23 Then, in the following experiments, FcR blocking and PI staining were omitted. As negative controls, cells were left unstained.

Wada N., Ikeda J., Nojima S., Tahara S., Ohshima K., Okuzaki D, & Morii E. (2016). Requirement of CXCL12-CXCR7 signaling for CD20(-) CD138(-) double-negative population in lymphoplasmacytic lymphoma. Laboratory investigation; a journal of technical methods and pathology, 96(5).

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Generating Mast Cell Precursors from CD34+ Cells

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Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Etablissement Français du Sang). CD34⁺ precursors cells were isolated from PBMCs (EasySep™ Human CD34 Positive Selection Kit, STEMCELL Technologies) and grown under serum-free conditions using StemSpanTM medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/ml; Peprotech), human IL-3 (10 ng/ml; Peprotech) and 3% supernatant of CHO transfectants secreting murine SCF (a gift from Dr. P. Dubreuil, Marseille, France, 3% correspond to ∼50 ng/ml SCF) for 1 week. Cells were next grown in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, .5% BSA, Insulin-transferrin selenium (all from Invitrogen), penicillin (100 U/ml), streptomycin (100 μg/ml) and 3% supernatant of CHO transfectants secreting murine SCF for 8 weeks then tested phenotypically (CD117+, FcεRI+) and functionally (β-hexosaminidase release in response to FcεRI crosslinking) before use for experiments. Only primary cell lines showing more than 95% CD117+/FcεRI+ cells were used for experiments.

Ramos-Llorca A., Decraecker L., Cacheux V.M., Zeiburlina I., De bruyn M., Battut L., Moreno-Cinos C., Ceradini D., Espinosa E., Dietrich G., Berg M., De Meester I., Van Der Veken P., Boeckxstaens G., Lambeir A.M., Denadai-Souza A, & Augustyns K. (2023). Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases. Frontiers in Chemistry, 10, 1089959.

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Differentiation of Human Mast Cells from PBMCs

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Human peripheral blood mononuclear cell-derived mast cells were generated as previously described by Gaudenzio et al.(73 (link)). Briefly, peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors and CD34+ precursor cells were isolated using the EasySep Human CD34 Positive Selection Kit (STEMCELL Technologies). CD34+ cells were maintained for 4 weeks under serum-free conditions using StemSpan medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/ml; Peprotech), human IL-3 (10 ng/ml; Peprotech) and human Stem Cell Factor (100 ng/mL Peprotech, Rocky Hill, NJ). Thereafter, the cells were maintained in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, insulin- 175 transferrin selenium (all from Invitrogen), ciprofloxacin (10 ug/ml; Sigma-Aldrich), IL-6 (50 ng/ml) and human Stem Cell Factor (100 ng/mL Peprotech, Rocky Hill, NJ). After 8-12 weeks, PBCMCs were tested for maturity by Giemsa or toluidine blue staining and beta-hexosaminidase release assays (see below).

Folkerts J., Redegeld F., Folkerts G., Blokhuis B., van den Berg M.P., de Bruijn M.J., van IJcken W.F., Junt T., Tam S.Y., Galli S.J., Hendriks R.W., Stadhouders R, & Maurer M. (2020). Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling. Allergy, 75(8), 1966-1978.

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Isolation and Culture of Human Mast Cells

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Human PBCMCs were obtained as previously described (Gaudenzio et al., 2016 (link)). Briefly, peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors at the Stanford Blood Center. CD34⁺ precursor cells were isolated from peripheral blood mononuclear cells (EasySep Human CD34 Positive Selection Kit, STEMCELL Technologies). CD34⁺ cells were maintained for 1 week under serum-free conditions using StemSpan medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/ml; Peprotech), human IL-3 (10 ng/ml; Peprotech) and 3% supernatant of CHO transfectants secreting murine SCF (a gift from Dr P. Dubreuil, Marseille, France, 3% correspond to ~50 ng/ml SCF). Thereafter, the cells were maintained in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, insulin-transferrin selenium (all from Invitrogen), ciprofloxacin (10 mg/ml; Sigma-Aldrich), IL-6 (50 ng/ml) and 3% supernatant of CHO transfectants secreting mouse SCF. Before use in experiments, PBCMCs were tested for phenotype by flow cytometry (tryptase, chymase, CD117, FcεRI) and function (β-hexosaminidase release in response to FcεRI cross-linking) at 8–12 weeks. PBCMCs were ready for experiments after ~10 weeks in culture, at which point mast cells (CD117⁺FcεRI⁺) represented ~95–99% of all cells.

Ho C.C., Chhabra A., Starkl P., Schnorr P.J., Wilmes S., Moraga I., Kwon H.S., Gaudenzio N., Sibilano R., Wehrman T.S., Gakovic M., Sockolosky J.T., Tiffany M.R., Ring A.M., Piehler J., Weissman I.L., Galli S.J., Shizuru J.A, & Garcia K.C. (2017). Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling. Cell, 168(6), 1041-1052.e18.

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Generation of Mast Cells from CD34+ Cells

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Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Etablissement Français du Sang). CD34⁺ precursors cells were isolated from PBMCs (EasySep™ Human CD34 Positive Selection Kit, STEMCELL Technologies). CD34⁺ cells were grown under serum-free conditions using StemSpan™ medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng.mL^-1; Peprotech), human IL-3 (10 ng.mL^-1; Peprotech) and 3% supernatant of CHO transfectants secreting murine SCF (a gift from Dr. P. Dubreuil, Marseille, France, 3% correspond to ~50 ng.mL^-1 SCF) for one week. Cells were grown in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, Insulin-transferrin selenium (all from Invitrogen), ciprofloxacin (10 µg.mL^-1; Sigma Aldrich), IL-6 (50 ng.mL^-1) and 3% supernatant of CHO transfectants secreting murine SCF for 8 weeks and tested both phenotypically (Tryptase⁺, CD117⁺, FcεRI⁺) and functionally (β-hexosaminidase release in response to FcεRI crosslinking) before use in experiments. Only primary cell lines showing more than 95% CD117⁺/FcεRI⁺ cells were used for experiments.

Eliasse Y., Leveque E., Garidou L., Battut L., McKenzie B., Nocera T., Redoules D, & Espinosa E. (2021). IL-17+ Mast Cell/T Helper Cell Axis in the Early Stages of Acne. Frontiers in Immunology, 12, 740540.

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