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Methocult gf m3534

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MethoCult GF M3534 is a methylcellulose-based medium formulated for the culture of human and mouse hematopoietic progenitor cells. It supports the growth and differentiation of granulocyte, erythroid, macrophage, and megakaryocyte colonies.

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Lab products found in correlation

Crystal violet, by Merck Group (1 mentions) Cfu gm, by STEMCELL (1 mentions) L butionine sulfoxamine bso, by Merck Group (1 mentions) Bz x700 microscope, by Keyence (1 mentions) Methocult gf m3434, by STEMCELL (1 mentions) Murine thrombopoietin tpo, by Thermo Fisher Scientific (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Lympholyte m, by Cedarlane (1 mentions) Macs separation ms columns, by Miltenyi Biotec (1 mentions) Il 11, by R&D Systems (1 mentions) Flt3 ligand, by Thermo Fisher Scientific (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions)

13 protocols using methocult gf m3534

1

Culturing CD45-/CD31+ Hematopoietic Cells

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The CD45−/CD31+ LSP and LMP cells (7,000 cells/ml) were cultured in Methocult GF M3534 medium according to the manufacturer's protocol (StemCell Technologies, Inc., Vancouver, Canada) as described previously (17 (link),19 (link),27 (link),28 (link)). Cell colonies, consisting of >30 cells, were scored after 14 days in culture. The cell colonies were fixed with 4% paraformaldehyde and stained with crystal violet based on the manufacturer's protocol (Sigma-Aldrich; Merck KGaA). Photomicrographs were obtained, from which the colonies were counted using an Olympus-DP70 microscope and images were captured with a digital camera (BX51). In order to determine whether the cells retained their CD45−/CD31+ LSP phenotype, Methoult medium was cut into sections and these were incubated with DMEM at 37°C with agitation for 30 min. When the Methocult medium had dissolved in the DMEM, the cells were collected, labeled with the CD45-PerCP-Cy5.5 (1:100), CD31-PE (1:100) and SCA1-FITC (1:100) antibodies, and analyzed by FACS.
Xu Y., Sun P., Wang J.Y., Li Z.Z., Gao R.L., Wang X.Z., Phillips W.D, & Liang S.X. (2019). Differentiation of CD45−/CD31+ lung side population cells into endothelial and smooth muscle cells in vitro. International Journal of Molecular Medicine, 43(3), 1128-1138.
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2

Myeloid Progenitor Colony Assay

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5x103 BM cells were suspended in methocult medium (MethoCult GF M3534; Stem Cell Technologies) with specific cytokines to promote the growth of myeloid progenitors. Colonies were counted after 10-12 days. Cells were collected by washing with PBS and stained for CD11b and cKit.
Khan N., Downey J., Sanz J., Kaufmann E., Blankenhaus B., Pacis A., Pernet E., Ahmed E., Cardoso S., Nijnik A., Mazer B., Sassetti C., Behr M.A., Soares M.P., Barreiro L.B, & Divangahi M. (2020). M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity. Cell, 183(3), 752-770.e22.
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3

CFU-GM Assay of Bone Marrow Cells

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Bone marrow cells (2×104) from WT or CGD mice were seeded in semisolid Methocult GF M3534 medium containing rmSCF, rmIL-3 and rhIL-6 for detection of CFU-GM (Stem Cell Technologies). L-butionine-sulfoxamine (BSO, Sigma-Aldrich) was added to methylcellulose media at the indicated concentrations at the time of plating.
Kwak H.J., Liu P., Bajrami B., Xu Y., Park S.Y., Nombela-Arrieta C., Mondal S., Sun Y., Zhu H., Chai L., Silberstein L.E., Cheng T, & Luo H.R. (2015). Myeloid cell-derived reactive oxygen species externally regulate the proliferation of myeloid progenitors in emergency granulopoiesis. Immunity, 42(1), 159-171.
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4

Expansion and Maintenance of Mouse Hematopoietic Cells

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Transduced mouse bone marrow stem and progenitor cells were expanded by serial plating in growth-factor containing semisolid medium (MethoCult GF M3534, Stem Cell Technologies, Vancouver, Canada) for four rounds at 5% CO2 at 37°C. Next the cells were harvested and maintained in liquid cultures in medium containing IL3, IL6 and mSCF as described previously [32 (link)].
Fahrenkrog B., Martinelli V., Nilles N., Fruhmann G., Chatel G., Juge S., Sauder U., Di Giacomo D., Mecucci C, & Schwaller J. (2016). Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype. PLoS ONE, 11(3), e0152321.
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5

Evaluating Hematopoietic Stem Cell Colony Formation

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Two hundred LKS+ cells were isolated from WT or STING−/− mice and were cultured with or without 50 μg/ml SP-derived EVs in 1.1 ml of Methocult GF M3434 (STEMCELL technologies, Vancouver, BC, Canada). The number of colonies and total cells was determined 7 and 10 days after incubation, respectively. In some experiments, 200 LKS+ cells or 1,250 mouse AML cells were cultured with or without 5 μM SBI-0206965 in 1.1 ml of Methocult GF M3534 (STEMCELL technologies). The number of colonies and total cells was determined 7 days after incubation. A wide-area continuous image of colony formation in a 35 mm dish was obtained using BZ-X700 microscope (Keyence, Osaka, Japan) and was analyzed using Keyence Analysis Software.
Baba T., Yoshida T., Tanabe Y., Nishimura T., Morishita S., Gotoh N., Hirao A., Hanayama R, & Mukaida N. (2021). Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway. Cell Death & Disease, 12(4), 322.
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6

AML Transformation Assay using Leukemic Cells

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Cells were plated in MethoCult GF M3534 (STEMCELL Technologies, Inc.). Colony numbers were counted and cells were collected, pooled, and replated at the indicated cell numbers per 35‐mm plate. For AE and AE9a colony replating assay, c‐Kit+ bone marrow (BM) cells were isolated by magnetic‐activated cell sorting (MACS) (Miltenyi Biotec) and then cultured in medium containing 100 ng/ml murine stem cell factor (SCF), 100 ng/ml murine thrombopoietin (TPO) and 20 ng/ml murine interleukin‐6 (IL‐6) (Peprotech). The cells were subsequently infected with a retrovirus vector (MigR1‐AE, Addgene plasmid #12431; MigR1‐AE9a, Addgene plasmid #12433, respectively) twice, with a 24‐h interval. Flow cytometry‐sorted EGFP+ cells were used for further experiments. For RNA‐sequencing (RNA‐seq), RNAs were extracted from colony forming cells of the first plating round.
Ueda T., Kanai A., Komuro A., Amano H., Ota K., Honda M., Kawazu M, & Okada H. (2021). KDM4B promotes acute myeloid leukemia associated with AML1‐ETO by regulating chromatin accessibility. FASEB BioAdvances, 3(12), 1020-1033.
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7

Quantification of CFU-GM Assay

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BM-MNCs were obtained by using Ficoll reagent and either ten or five thousand cells were tested for CFU-GM assay in a 30 mm culture dish by using Methocult medium (Methocult GF M3534, Stem Cell Technologies) as per manufacturer’s protocol. After 10 days, colonies were counted, and bright field images were taken by using a microscope (Leica). Then, colonies were dissociated, and inflammatory cells were enumerated by flow cytometry as described above. Where applicable, antagonist or vehicle was added to the cell-suspension prior to mixing with Methocult.
Chittimalli K., Jahan J., Sakamuri A., Weyrick H., Winkle W., Adkins S., Vetter S.W, & Jarajapu Y.P. (2023). Reversal of aging-associated increase in myelopoiesis and expression of alarmins by angiotensin-(1–7). Scientific Reports, 13, 2543.
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8

Isolation and Characterization of Hepatic Leukocytes

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Freshly isolated livers were minced into small pieces, treated with collagenase-IV (17104-019, Gibco) and DNAse I (D5025, Sigma), and filtered through cell dissociation sieve to prepare a single cell suspension. Hepatic leukocytes (WBCs) were isolated using mouse cell separation media, Lympholyte M (CL5035, Cedarlane). Then the total hepatic WBC count was analyzed using vet ABC animal blood counter and data presented as WBC count/gram of the liver. For flow cytometry, isolated leukocytes were stained with the indicated antibodies for 30 minutes at 4°C. The antibodies used were anti-CD45-V450 (clone 30-F11, BD Pharmingen) anti-CD201-APC (clone ebio1560, ebioscience) and anti-CD27-BV650 (clone LG3A10, BD Pharmingen). Flow cytometry was performed on BD LSRFortessa ×50 platforms and results were analysed using FlowJo software version 9.9.5 (TreeStar). For CFU-GM assay, 105 cells were cultured in 1.1 ml of methylcellulose-based medium for myeloid progenitor cells (MethoCult™ GF M3534, STEM cell technologies) as per company protocol. Colonies of at least 50 cells were scored for analysis of CFU-GM.
Khadge S., Sharp J.G., Thiele G.M., McGuire T.R., Klassen L.W., Duryee M.J., Britton H.C., Dafferner A.J., Beck J., Black P.N., DiRusso C.C, & Talmadge J. (2017). Dietary Omega-3 and Omega-6 Polyunsaturated Fatty Acids Modulate Hepatic Pathology. The Journal of nutritional biochemistry, 52, 92-102.
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9

Bone Marrow Colony-Forming Assay

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20,000 bone marrow cells were plated per mL of Methocult GF M3534 in triplicate for each biological sample (Stemcell Technologies, Vancouver, BC, Canada). Colonies were counted 12 days later.
Grigsby S.M., Friedman A., Chase J., Waas B., Ropa J., Serio J., Shen C., Muntean A.G., Maillard I, & Nikolovska-Coleska Z. (2021). Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis. Cancers, 13(4), 642.
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10

Isolation and Culture of Hematopoietic Progenitors

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Haematopoietic progenitors were isolated from the BM of DsRed+Wt and Map3k8−/− mice by magnetic-activated cell sorting (MACS) using a LIN− lineage depletion kit (containing antibodies against CD5, CD11b, GR-1, 7-4, and Ter119) and MACS separation MS columns (Miltenyi). Isolated LIN− DsRed+Wt and Map3k8−/− cells (2,500) were plated at a 1:1 ratio in a well with methylcellulose medium, which contains insulin, H transferrin, stem cell factor, IL-3, and IL-6 (MethoCult GF M3534, StemCell Technologies) in the presence or absence of LPS (500 ng/ml). After six days of incubation at 37 °C in an atmosphere containing 5% CO2, the cells were harvested and subjected to FACS analysis with Perfect-Count microspheres.
Sánchez Á., Relaño C., Carrasco A., Contreras-Jurado C., Martín-Duce A., Aranda A, & Alemany S. (2017). Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis. Scientific Reports, 7, 5010.
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