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Methocult m3234

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MethoCult M3234 is a methylcellulose-based medium used for the culture and enumeration of mouse and human hematopoietic progenitor cells. The medium supports the growth and differentiation of various blood cell lineages from these progenitor cells.

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Lab products found in correlation

Methocult m3434, by STEMCELL (4 mentions) Stem cell factor (scf), by STEMCELL (3 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions) Ckx31 inverted microscope, by Olympus (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Methocult h4034 optimum, by STEMCELL (2 mentions) Benzidine solution, by Merck Group (2 mentions) Megacult, by STEMCELL (2 mentions) Dulbecco modified eagle medium (dmem), by STEMCELL (2 mentions) Lineage cell depletion kit, by Miltenyi Biotec (2 mentions) B6.129s4 il2rgtm1wjl j mice, by Jackson ImmunoResearch (2 mentions) Wehi 3b, by STEMCELL (2 mentions) Anti human il2rγ pe, by BioLegend (2 mentions) Murine il 3, by STEMCELL (2 mentions) Interleukin 6 (il 6), by STEMCELL (2 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Dmi4000b, by Leica (1 mentions) Interleukin 6 (il 6), by GoldBio (1 mentions) Interleukin 3 (il 3), by GoldBio (1 mentions)

Spelling variants of this product

MethoCult (224 citations) M3234 (18 citations) MethoCult GF M3234 (3 citations) MethoCult M3234 Methylcellulose medium (2 citations)

32 protocols using methocult m3234

1

Erythroid Progenitor Quantification

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Colony assays were performed as described previously (29 (link)–31 (link)). For CFU-E analysis, nucleated bone marrow (1×105/mL) and splenocytes (2×106/mL) were plated onto Methocult M3234 (Stem Cell Technologies) containing 3U/mL EPO (Peprotech) and placed into a humidified chamber under normoxia (21% O2). The presence of small, rose-colored colonies were counted 2 days after plating using an inverted microscope.
Mumau M.D., Vanderbeck A.N., Lynch E.D., Golec S.B., Emerson S.G, & Punt J.A. (2017). Identification of a multipotent progenitor population in the spleen that is regulated by NR4A1. Journal of immunology (Baltimore, Md. : 1950), 200(3), 1078-1087.
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2

Hematopoietic Progenitor Colony Assay

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Sorted pre-MegEs and MkPs were seeded at 500 cells/dish of 35-mm colony dishes (STEMCELL Technologies) in Methocult M3234 (STEMCELL Technologies) supplemented with 25 ng/ml SCF, 10 ng/ml IL-3, 20 ng/ml IL-11, 10 ng/ml GM-CSF, 3 U/ml Epo, and 10 ng/ml Tpo. Colonies were scored between 7 and 10 d using an inverted microscope (DMI 4000B; Leica).
Foudi A., Kramer D.J., Qin J., Ye D., Behlich A.S., Mordecai S., Preffer F.I., Amzallag A., Ramaswamy S., Hochedlinger K., Orkin S.H, & Hock H. (2014). Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation. The Journal of Experimental Medicine, 211(5), 909-927.
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3

Leukemic Cell Clonogenic Potential

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Wild-type or HMGN1-OE cells stably infected with the oncogenes indicated were seeded in methylcellulose media (Methocult M3234, Stem Cell Technologies), supplemented with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml) (GoldBio) at 2 × 105 cells/ml and at 5 × 104 cells/ml in subsequent passages. Colonies were manually counted at 7 days, pooled, and replated. For long-term culture initiating colony (LTC-IC) assays, leukemic splenocytes from three different animals were plated on MS5 feeder cells for three weeks in RPMI media enriched with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml). Cells were counted and 10,000, 20,000, or 50,000 GFP+ cells were seeded in methylcellulose media (Methocult M3234) supplemented with SCF (10 ng/ml), IL-3 (6 ng/ml), and IL-6 (10 ng/ml) in 35 mm dishes. One week later, the number of colonies and cells was determined.
Cabal-Hierro L., van Galen P., Prado M.A., Higby K.J., Togami K., Mowery C.T., Paulo J.A., Xie Y., Cejas P., Furusawa T., Bustin M., Long H.W., Sykes D.B., Gygi S.P., Finley D., Bernstein B.E, & Lane A.A. (2020). Chromatin accessibility promotes hematopoietic and leukemia stem cell activity. Nature Communications, 11, 1406.
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4

Generation of Murine and Human Leukemia Models

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BM cells were obtained from femoral and tibial bones, and stained with Lineage-Biotin (559971, BD Biosciences), Sca-1-PE (122508, BioLegend), c-KIT-Alexa Flour 647 (105818, BioLegend) antibodies and Streptoavidin-FITC (554060, BD Biosciences), then LSK cells were isolated with FACS Aria (BD Biosciences). LSK cells were cultured in RPMI medium 1640 supplemented with 10% FBS, 20 ng/ml rmSCF, 10 ng/ml rmIL3, 10 ng/ml rmIL6. After 24 h of cultivation, spin infection was performed by adding retrovirus carrying MLL-AF9-ires-Neo or MLL-AF9-ires-GFP in the media containing 8 μg/ml polybrene to LSK cells and centrifuging at 1,400 × g for 90 min. MLL-AF9 expressing LSK cells were serially re-plated with methylcellulose medium (STEMCELL Technologies, MethoCult M3234) containing the above cytokines for 3 weeks. To obtain the fully transformed leukemia cells, immortalized cells were transplanted into lethally irradiated (9.5 Gy) syngeneic recipient mice along with 5 × 105 bone marrow cells from wild-type mice. Human cord blood CD34+ cells were enriched by microbeads (Miltenyi Biotec) and MLL-AF9 transduced cells were transplanted to NOD/SCID mice. AML cells were harvested from bone marrow or spleen and cultured in IMDM medium supplemented with 20% FBS, beta-mercaptoethanol, 20 ng/ml rhIL6/rhTPO/rhSCF/rhGM-CSF, 10 ng/ml IL3.
Hoshii T., Cifani P., Feng Z., Huang C.H., Koche R., Chen C.W., Delaney C.D., Lowe S.W., Kentsis A, & Armstrong S.A. (2018). A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response. Cell, 172(5), 1007-1021.e17.
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5

Murine BMMC Colony Forming Assay

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Upon initial plating, a total of 1×104 murine BMMC cells per well were cultured in 1mL of methylcellulose medium (Methocult M3234, STEMCELL Tech.) containing 50ng/ml SCF, 10ng/ml IL-3, 10ng/ml IL-6 and 3U/ml Erythropoietin (EPO, Catalog # GH002, HumanCells Bio.) and 1×penicillin and streptomycin in 6-wells plate. After incubation at 37°C under 5% CO2 and high humidity for 14 days, colony-forming units (CFUs) (>50 cells) were scored according to the manufacturer’s instructions under Olympus CKX31 inverted microscope. For serial re-plating experiments, the cells from colonies were pooled and dissociated into single cells by pipetting with PBS. 1×104 cells per well were further seeded for another 14 days. To qualify colony size, the colonies were photographed by Olympus IX51 with cellSens Entry software. The colony area was calculated by Image J (1.45s).
Li Z., Su M., Xie X., Wang P., Bi H., Li E., Ren K., Dong L., Lv Z., Ma X., Liu Y., Zhao B., Peng Y., Liu J., Liu L., Yang J., Ji P, & Mei Y. (2023). mDia formins form hetero-oligomers and cooperatively maintain murine hematopoiesis. PLOS Genetics, 19(12), e1011084.
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6

Murine Bone Marrow Colony Formation

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BM stem cells (2x104) from Zhx2 wt and null mice were cultured in 35mm dish with IMDM (Thermo Fischer Scientific), consisting MethoCult M3234 (Stemcell Technologies) and M-CSF (30ng/ml). Media were changed every 2 days. After twelve days we counted the number of colonies (50 cells or more) in each genotype 15 (link).
Erbilgin A., Seldin M.M., Wu X., Mehrabian M., Zhou Z., Qi H., Dabirian K.S., Sevag Packard R.R., Hsieh W., Bensinger S.J., Sinha S, & Lusis A.J. (2018). Transcription factor Zhx2 deficiency reduces atherosclerosis and promotes macrophage apoptosis in mice. Arteriosclerosis, thrombosis, and vascular biology, 38(9), 2016-2027.
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7

Methylcellulose Assay for Hematopoietic Progenitors

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We plated 5,000 cells from each population in 1.5 ml of Methocult M3234 (Stem Cell Technologies) supplemented with IL3 (10 ng/ml), IL6 (5 ng/ml), IL7 (10 ng/ml) and stem cell factor (20 ng/ml). Methylcellulose cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 in air. Colonies were scored on day 7.
Saadatpour A., Guo G., Orkin S.H, & Yuan G.C. (2014). Characterizing heterogeneity in leukemic cells using single-cell gene expression analysis. Genome Biology, 15(12), 525.
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8

Hematopoietic Stem Cell Culture Conditions

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For growth assays, sorted CD34-CD150+LSK HSCs were cultured in S-Clone SF-O3 (Sanko Junyaku) supplemented with 0.1% BSA (093001; STEMCELL Technologies), 50 μM 2-ME (Sigma-Aldrich) and 1% penicillin/streptomycin/glutamine (Gibco). 20 ng/mL of recombinant mouse SCF (579706; BioLegend) and recombinant human TPO (763706; BioLegend) for HSC culture conditions and 10 ng/mL of SCF, TPO, recombinant mouse IL-3 (575506; BioLegend), and recombinant murine GM-CSF (315-03; PeproTech) for myeloid culture condition-1 were added to cultures. In the case of myeloid culture condition-2, sorted CD150+CD48-CD135-CD34-LSK HSCs, LSK cells, and GMPs were cultured in IMDM (Gibco) supplemented with 5% FBS, 50 μM 2-ME (Sigma-Aldrich), 1% penicillin/streptomycin/glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 0.1 mM MEM Non-Essential Amino Acids solution (Gibco). 25 ng/mL of SCF, TPO, recombinant human Flt3L (300-19; PeproTech) and recombinant murine IL-11 (220-11; PeproTech) and 10 ng/mL of IL-3 and GM-CSF were added to cultures.
For replating assays, LSK cells were plated in methylcellulose medium (Methocult M3234; STEMCELL Technologies) containing 20 ng/mL of SCF, TPO, IL-3, and GM-CSF.
Nakajima-Takagi Y., Oshima M., Takano J., Koide S., Itokawa N., Uemura S., Yamashita M., Andoh S., Aoyama K., Isshiki Y., Shinoda D., Saraya A., Arai F., Yamaguchi K., Furukawa Y., Koseki H., Ikawa T, & Iwama A. (2023). Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis. eLife, 12, e83004.
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9

Enumerating Cytokine-independent Hematopoietic Colonies

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Epo-independent endogenous CFU-E and cytokine-independent CFU-G were enumerated in methylcellulose media without exogenous cytokines (MethoCult M3234, StemCell Technologies). Sorted CMPs were plated with IL3, IL6, SCF, and Epo (MethoCult M3434, StemCell Technologies). Colonies were scored according to manufacturer manual.
Yao H., Ma Y., Hong Z., Zhao L., Monaghan S.A., Hu M.C, & Huang L.J. (2017). Activating JAK2 mutants reveal cytokine receptor coupling differences that impact outcomes in myeloproliferative neoplasm. Leukemia, 31(10), 2122-2131.
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10

MLL-AF9-Induced Leukemogenesis Assay

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Lin-BM cells were isolated and transduced with recombinant MLL-AF9 using published protocols [17 (link), 29 (link)]. Cells (0.5 × 104) were plated in methylcellulose media (MethoCult™ M3234, STEMCELL Technologies), and replating was performed every 7–10 days. CFU (with > 50 cells) was scored during each round of plating. For subsequent cell culture, individual colonies were plucked using a P200 micropipettor and transferred to microcentrifuge tubes containing 500 μl of PBS. Pelleted cells were then resuspended in BM culture media [17 (link)] and maintained in a humid 37 °C 5%CO2-incubator. For in vitro recombination, 4-OHT (Sigma-Aldrich) was resuspended in ethanol and added to cell culture at final concentration of 250 nM. Media was changed daily during culture. For in vivo transplantation, MLL-AF9-transduced cells (5 × 105) were injected through a tail vein into lethally irradiated (900 cGY) mice. Recipient mice were maintained on antibiotics for 2 weeks.
Yang L., Liu L., Gao H., Pinnamaneni J.P., Sanagasetti D., Singh V.P., Wang K., Mathison M., Zhang Q., Chen F., Mo Q., Rosengart T, & Yang J. (2017). The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis. Journal of Hematology & Oncology, 10, 159.
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