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Methocult m3231

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Methocult M3231 is a methylcellulose-based medium used for the in vitro culture of human hematopoietic progenitor cells. It supports the growth and differentiation of colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) colonies from human hematopoietic stem and progenitor cells.

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Lab products found in correlation

Methocult h4230, by STEMCELL (3 mentions) Methocult m3434, by STEMCELL (3 mentions) Rmgm csf, by R&D Systems (2 mentions) Gm csf, by Thermo Fisher Scientific (2 mentions) Murine gm csf, by Thermo Fisher Scientific (2 mentions) Macs ls column, by Miltenyi Biotec (2 mentions) Rmscf, by R&D Systems (1 mentions) Iscove s modified dulbecco medium, by Lonza (1 mentions) Dmi8 inverted microscope, by Leica (1 mentions) Gm csf, by STEMCELL (1 mentions) Celltiter 96 aqueous one solution reagent, by Promega (1 mentions) M3534, by STEMCELL (1 mentions) H4434, by STEMCELL (1 mentions) H4230, by STEMCELL (1 mentions) Histopaque, by Merck Group (1 mentions) Cd3 apc, by BioLegend (1 mentions) Cd11b percp cy5, by BD (1 mentions) Cd19 pe, by Thermo Fisher Scientific (1 mentions) Cd45 apc cy7, by BioLegend (1 mentions) Facsverse, by BD (1 mentions)

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M3231 MethoCult

30 protocols using methocult m3231

1

Murine Hematopoietic Cell Culture

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THP-1 cells were cultured at 500,000 cells/ml in RPMI-1640 GlutaMAX containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. embryo (MLL-AF9 KI/+ mice 15 were obtained from The Jackson Laboratory). After c-Kit enrichment using MACS LS columns (Miltenyi Biotec), cells were serially replated every 6 d in MethoCult M3231
(STEMCELL Technologies) supplemented with 20ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6 and 10 ng/ml GM-CSF. After 3 rounds of plating, cells were cultured at 300,000 cells/ml in IMDM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, supplemented with SCF, IL-3, and IL-6.
MMH (Meis1/Hoxa9 cells): foetal liver haematopoietic cells were extracted from a E14.5 C57Bl/6 embryo. Following c-Kit enrichment using MACS LS columns (Miltenyi Biotec), cells were transduced with MSCV-Meis1a-puro and MSCV-Hoxa9-neo retroviruses as per 14 . Following selection for puromycin/neomycin co-resistance, cells were serially replated every 6 days in MethoCult M3231
(STEMCELL Technologies) supplemented with 20 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6 and 10 ng/ml GM-CSF. After 3 rounds of plating, cells were cultured at 200,000 cells/ml in IMDM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, supplemented with SCF, IL-3, and IL-6.
O'Duibhir E., Paris J., Lawson H., Sepulveda C., Shenton D.D., Carragher N.O, & Kranc K.R. (2018). Machine Learning Enables Live Label-Free Phenotypic Screening in Three Dimensions. Assay and drug development technologies, 16(1).
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2

Quantifying CFU-GM in Mouse Bone Marrow

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To detect colony-forming unit granulocyte monocyte (CFU-GM), 5 × 104 total bone marrow cells in 10% of the total volume were seeded in semisolid methylcellulose in 80% of the total volume (MethoCult M3231, Stem Cell Technology, France) with 10% of the total volume of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with rm-SCF (100 ng/mL), rm-interleukin-3 (30 ng/mL) and rm-GM-CSF (30 ng/mL) (R&D Systems). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2, and colonies were scored 7 days later under an inverted microscope.
Yadak R., Cabrera-Pérez R., Torres-Torronteras J., Bugiani M., Haeck J.C., Huston M.W., Bogaerts E., Goffart S., Jacobs E.H., Stok M., Leonardelli L., Biasco L., Verdijk R.M., Bernsen M.R., Ruijter G., Martí R., Wagemaker G., van Til N.P, & de Coo I.F. (2018). Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE. Molecular Therapy. Methods & Clinical Development, 8, 152-165.
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3

Myeloid and Erythroid Colony Assays

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For myeloid colonies, 1E4 total bone marrow cells were plated in 1 mL methylcellulose medium (Methocult M3231, Stem Cell Technologies, Vancouver, BC, Canada) with IMDM, 10% HI-FBS supplemented with 10 ng/mL murine IL-3, 10 ng/mL murine IL-6, and 50 ng/mL murine SCF. Colonies of at least 50 cells were counted between days 7 and 8. BFU-E were enumerated 8 days after 2E5 marrow cells per 1 mL were cultured in 40% MethoCult M3120, IMDM, 10% plasma-derived serum, 20% BIT (Stem Cell Technologies), 5% protein free hybridoma medium (PFHM), 2 mM glutamine, 55 nM β-mercaptoethanol, and 10 U/mL hEPO. For the serial replating assay, total colony cells were washed in sterile PBS and 1E4 cells in 1 mL methylcellulose medium were replated every 7 days for up to 6 rounds. A minimum of three independent experiments were performed in triplicates for each of the colony assays.
Zhang J., Li L., Baldwin AS J.r., Friedman A.D, & Paz-Priel I. (2015). Loss of IKKβ but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells. PLoS ONE, 10(6), e0130441.
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4

Clonogenic Assay for Cell Viability

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Clonogenic assay was realized with cells seeded into 35 mm petri dish in semi-solid methylcellulose medium (Methocult™ M3231 for murin cells or Methocult™ H4230 for human cells, Stemcell Technologies). Cells were treated with the drugs for 72 h and then centrifugated. The pellet was resuspended in Iscove's modified Dulbecco medium (Lonza) supplemented with 2% fetal calf serum and 50 U/ml penicillin, 50 mg/ml streptomycin at 10,000 cells/ml, and cell suspension was added to methylcellulose medium (1000 cells/1ml/dish) and left at 37 °C and 5% CO2. Colony forming efficiency was determined after 7 days using Leica DMI8 inverted microscope (Leica Microsystems) and quantified using Image J software.
Trinh A., Khamari R., Fovez Q., Mahon F.X., Turcq B., Bouscary D., Maboudou P., Joncquel M., Coiteux V., Germain N., Laine W., Dekiouk S., Jean-Pierre S., Maguer-Satta V., Ghesquiere B., Idziorek T., Quesnel B., Kluza J, & Marchetti P. (2021). Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells. Molecular Metabolism, 55, 101410.
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5

Isolation and Culture of Cardiac Myocytes

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Tyrode solution contained the following (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5.0 Hepes (pH adjusted to 7.4 with NaOH). The cell isolation buffer contained the following (in mM): 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 22 glucose, 50 μU mL−1 bovine insulin, and 25 Hepes (pH adjusted to 7.4 with NaOH). The cell isolation buffer was used to isolate both ventricular myocytes and ACMs. The cell suspension buffer contained Tyrode solution supplemented with 0.2 mg mL−1 bovine serum albumin (BSA), 100 μg mL−1 penicillin, 0.1 mg mL−1 streptomycin, and 15 μg mL−1 phenol red. The semisolid culture medium consisted of an 80:20 mixture of methylcellulose-based medium MethoCult® M3231 (STEMCELL Technologies) and Iscove’s modified Dulbecco’s medium (IMDM). The final composition of the cell culture medium was 1% methylcellulose, 30% fetal bovine serum (FBS), 1% BSA, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol in IMDM.
Omatsu-Kanbe M., Nozuchi N., Nishino Y., Mukaisho K.I., Sugihara H, & Matsuura H. (2017). Identification of cardiac progenitors that survive in the ischemic human heart after ventricular myocyte death. Scientific Reports, 7, 41318.
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6

Culturing Human and Mouse Leukemia Cells

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Human leukemia cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1× penicillin/streptomycin/L-glutamine (PSG), and 1× non-essential amino acids (NEAA). Transformed mouse myeloid cells or leukemia cells were cultured in RPMI 1640 medium supplemented with 20% FBS, 20% WEHI-conditioned medium, and PSG (R20/20 medium), or in methylcellulose-containing medium (Methocult M3231, Stem Cell Technologies) with cytokines as previously described (Lavau et al., 1997 (link)). HEK-293T and Phoenix-Eco cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and PSG.
Wong S.H., Goode D.L., Iwasaki M., Wei M.C., Kuo H.P., Zhu L., Schneidawind D., Duque-Afonso J., Weng Z, & Cleary M.L. (2015). The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential. Cancer cell, 28(2), 198-209.
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7

Assessing Spheroid Formation in AMs

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The method was performed as previously described, with minor modifications [29] (link), [30] . AMs were plated at 10,000 cells per 1 mL of Methocult M3231 (StemCell Technologies, Vancouver, BC, Canada) containing 100 ng/ml GM-CSF (Peprotech) and with-GABA (100 μM) or without-GABA, grown at 37 °C and 5% CO2. The spheroid-forming rate (SFR) was counted after 21 days.
Gao Y., Liang Z., Mao B., Zheng X., Shan J., Jin C., Liu S., Kolliputi N., Chen Y., Xu F, & Shi L. (2023). Gut microbial GABAergic signaling improves stress-associated innate immunity to respiratory viral infection. Journal of Advanced Research.
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8

Evaluating Colony Formation Capacity

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1×104 whole bone marrow cells isolated from pIpC-treated Kras+/G12D or wild-type mice were plated in 1.5ml of Methocult M3231 (Stemcell Technologies) supplemented with the indicated concentrations of GM-CSF (Peprotech) and RGS in a non-treated 35mm dish in duplicate. Cells were cultured for 7 days before colonies were scored.
Baker S.J., Cosenza S.C., Ramana Reddy M.V, & Premkumar Reddy E. (2019). Rigosertib ameliorates the effects of oncogenic KRAS signaling in a murine model of myeloproliferative neoplasia. Oncotarget, 10(20), 1932-1942.
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9

Hematopoietic Progenitor Assays for LZTR1 Mutants

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20×103 fetal liver cells from Lztr1+/+ and Lztr1−/− E14.5 embryos were seeded into cytokine-supplemented methylcellulose medium (Methocult M3434; STEMCELL Technologies). Colonies propagated in culture were scored at day 7. The remaining cells were resuspended and counted, and a portion was taken for replating every week.
To test GM-CSF hypersensitivity, LSK (Lineage-negative c-Kit+ Sca1+DAPI−) were FACS-sorted from the BM of Mx1-cre, Mx1-cre Lztr1fl/fl, and Mx1-cre Rit1M90I/WT mice 4 weeks after pIpC administration and seeded at a density of 800 cells/replicate into methylcellulose medium (Methocult M3231; STEMCELL Technologies) with murine GM-CSF (PeproTech) at dose of 0, 0.01, 0.1, 1, 10 ng/ml. Colonies were scored at day 7.
Chen S., Vedula R.S., Cuevas-Navarro A., Lu B., Hogg S.J., Wang E., Benbarche S., Knorr K., Kim W.J., Stanley R.F., Cho H., Erickson C., Singer M., Cui D., Tittley S., Durham B.H., Pavletich T.S., Fiala E., Walsh M.F., Inoue D., Monette S., Taylor J., Rosen N., McCormick F., Lindsley R.C., Castel P, & Abdel-Wahab O. (2022). Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation. Cancer Discovery, 12(10), 2434-2453.
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10

FIP1L1-PDGFRα Oncogene Cell Assays

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The EOL-1 cell line harboring the FIP1L1-PDGFRα fusion oncogene was purchased from DMSZ (Braunschweig, Germany). BaF3 cells expressing WT or T674I FIP1L1-PDGFRα were cultured as described previously [8 (link),9 (link)].
Cell viability was assessed by MTS assay (CellTiter 96 Aqueous One Solution reagent, Promega, Shanghai) [40 (link),42 (link)].
Clonogenicity assay was performed as described [40 (link)]. In brief, 2×105/ml cells were treated with drugs or diluent (DMSO, control) for 24 h, then washed with PBS and seeded in methylcellulose medium (Methocult M3231, Stem Cell Technologies, Vancouver, Canada) [40 (link)]. After incubation for ~7 days at 37°C and 5% CO2, colonies with >50 cells were counted [40 (link)].
Jin Y., Ding K., Li H., Xue M., Shi X., Wang C, & Pan J. (2014). Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and β-catenin. Molecular Cancer, 13, 17.
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