Methocult h4034 optimum

Manufactured by STEMCELL

Sourced in Canada

MethoCult H4034 Optimum is a semisolid medium used for the in vitro culture and differentiation of human and mouse hematopoietic progenitor cells. The medium contains essential growth factors and cytokines required for the growth and differentiation of various hematopoietic lineages, including erythroid, myeloid, and megakaryocytic cells.

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50 protocols using methocult h4034 optimum

Colony-Forming Cell Assay for CD34+ and CML Cells

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CD34⁺ cells (3 × 10³) were seeded in 1.5 ml of MethoCult H4034 Optimum (STEMCELL Technologies, #04034). For CML cell lines, 1 × 10³ cells were seeded in MethoCult 4230 (STEMCELL Technologies, #04230). The number of CFCs was counted after 14 days.

Ianniciello A., Zarou M.M., Rattigan K.M., Scott M., Dawson A., Dunn K., Brabcova Z., Kalkman E.R., Nixon C., Michie A.M., Copland M., Vetrie D., Ambler M., Saxty B, & Helgason G.V. (2021). ULK1 inhibition promotes oxidative stress–induced differentiation and sensitizes leukemic stem cells to targeted therapy. Science translational medicine, 13(613), eabd5016.

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Hematopoietic Progenitor Cell Characterization

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Primary AML (50 × 10³) or lineage-negative cord blood cells (1 × 10³) cells were treated for 18 h with bromocriptine and mixed with 1 mL of MethoCult H4034 Optimum (StemCell Technologies). Colonies were screened based on morphology and cellularity at day 14 by light microscopy. BFU-E (burst-forming unit-erythroid) contains >200 erythroblasts in a single or multiple clusters. CFU-GM (colony-forming unit-granulocyte/macrophage) is composed by at least 40 granulocytes and macrophages. When only granulocytes or macrophages are found, these colonies are identified as CFU-G and CFU-M, respectively. CFU-Mix or CFU-GEM is initiated by a multi-potential progenitor that produces a colony containing erythroblasts and cells of at least two other recognizable lineages. CFU-GEM tends to produce large colonies due to their primitive nature.

Lara-Castillo M.C., Cornet-Masana J.M., Etxabe A., Banús-Mulet A., Torrente M.Á., Nomdedeu M., Díaz-Beyá M., Esteve J, & Risueño R.M. (2016). Repositioning of bromocriptine for treatment of acute myeloid leukemia. Journal of Translational Medicine, 14(1), 261.

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In Vitro Colony-Forming Assay

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On day 8 of in vitro culture, cells were harvested and resuspended in IMDM. Indicated numbers of cells were transferred to 1 mL of MethoCult H4034 Optimum (STEMCELL Technologies, Vancouver, Canada). The mixture was then transferred to ultra-low attachment 24-well plates (Corning). Cells were cultured at 37 ℃ in 5% CO₂ with 100% humidity for 14 days. The plates were then visually scored for colony-forming unit-granulocyte/macrophage (CFU-GM), colony-forming unit-erythrocyte (CFU-E), burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte (CFU-G), colony-forming unit-macrophage (CFU-M), and colony-forming unit granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM).

Hua J., Jiao T., Qiao Y., Zhang S., Xiao T., Zhang Y, & Yan J. (2023). Human serum albumin promotes self-renewal and expansion of umbilical cord blood CD34+ hematopoietic stem/progenitor cells. Annals of Translational Medicine, 11(2), 62.

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Hematopoietic Colony Forming Assay

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Primary cells were plated in SFM supplemented with PGF cocktail in the
presence of indicated drugs. After 3 days, cells from each condition were
transferred in methylcellulose-based medium (Methocult H4034 Optimum, StemCell
Technologies) in duplicate and colonies were manually counted after 12-14
days.

Kuntz E.M., Baquero P., Michie A.M., Dunn K., Tardito S., Holyoake T.L., Helgason G.V, & Gottlieb E. (2017). Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemic stem cells. Nature medicine, 23(10), 1234-1240.

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Hematopoietic Progenitor Cell Enumeration

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Whole blood from each data point was plated for cultures of HPCs following Stemcell Technologies MethoCult protocol.^{18 (link)} Briefly, mononuclear cells in the peripheral blood were separated by Ficoll-Hypaque density gradient (Sigma-Aldrich, St. Louis, MO) then resuspended in IMDM + glutamine (Gibco Life Technologies, Grand Island, NY) containing 10% fetal calf serum (FCS, Hyclone Laboratories, Greeley, CO). The number of mononuclear cells was enumerated using an inverted microscope and then plated (1 × 10⁶ cells/mL) in MethoCult H4034 Optimum (Stemcell Technologies, Tukila, WA). Cultures were incubated at 37°C in 5% CO₂. Colonies (clusters of > 10 cells) were enumerated at the end of incubation (Day 14 for BFU-E and Day 7 for CFU-E) by an observer blinded to the origin of the samples.

Bible L.E., Pasupuleti L.V., Alzate W.D., Gore A.V., Song K.J., Sifri Z.C., Livingston D.H, & Mohr A.M. (2014). Early propranolol administration to severely injured patients can improve bone marrow dysfunction. The journal of trauma and acute care surgery, 77(1), 54-60.

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Hematopoietic Colony-Forming Unit Assay

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To assess hematopoietic colony forming unit (CFU) potential, 2 × 10⁴ to 1 × 10⁵ viable cells were plated in individual 35 mm dishes (StemCell Tech., Vancouver, CA; #27150) in 1.5 ml multi‐lineage CFU semi‐solid media (StemCell Technologies, Vancouver, CA; MethoCult H4034 Optimum) per dish. Duplicate dishes were generated for each condition. Colonies were scored after 14 days of culture at 37°C in normoxic, 5% CO₂ conditions. CFU values were normalized to the number of cells plated (CFU per 10⁴ cells plated).

Leung A., Zulick E., Skvir N., Vanuytsel K., Morrison T.A., Naing Z.H., Wang Z., Dai Y., Chui D.H., Steinberg M.H., Sherr D.H, & Murphy G.J. (2018). Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells. Stem Cells (Dayton, Ohio), 36(7), 1004-1019.

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Clonogenic Assay for Hematopoietic Cells

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Clonogenicity of surviving cells was assayed in semi-solid culture medium (MethoCult H4034 Optimum, Stem Cell Technologies) for 12 days. Briefly, the methylcellulose was seeded with a fixed volume or 3,000 viable cells and plated onto 35 mm culture dishes. Colonies were counted and scored as colony forming unit (CFU)-erythroid (E), CFU-granulocyte, -macrophage, -granulocyte/macrophage (G, M, G/M), CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) or burst forming unit-erythroid (BFU-E).

Laidlaw K.M., Berhan S., Liu S., Silvestri G., Holyoake T.L., Frank D.A., Aggarwal B., Bonner M.Y., Perrotti D., Jørgensen H.G, & Arbiser J.L. (2016). Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia. Oncotarget, 7(32), 51651-51664.

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Clonogenic Capacity of HD-CD34+ Cells

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The clonogenic capacity of HD-CD34⁺ cells, expanded after 8 days of co-culture with BM-MSC silenced for FOXM1 or in the control condition, was evaluated by short-term CFU assay. Briefly, after 8 days of coculture, HD CD34⁺ cells were seeded in triplicate in a 35 mm dish (at the final concentrations of 2,5 × 10³ cells) and cultured in MethoCult™ H4034 Optimum (StemCell Technologies) at 37 °C, in humidified atmosphere and 5% CO₂ for 12–14 days. After 12–14 days, the number of erythroid burst-forming unit (BFU-E), Colony Forming Unit-Erythroid (CFU-E), granulocyte–macrophage colony forming unit (CFU-GM) was determined with manual counts using an optical microscope. Biological and technical triplicate experiments were performed.

Falconi G., Galossi E., Fabiani E., Pieraccioli M., Travaglini S., Hajrullaj H., Cerretti R., Palmieri R., Latagliata R., Maurillo L, & Voso M.T. (2022). Impairment of FOXM1 expression in mesenchymal cells from patients with myeloid neoplasms, de novo and therapy-related, may compromise their ability to support hematopoiesis. Scientific Reports, 12, 21231.

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Colony Forming Unit Assay

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Single cells of the indicated numbers in 0.1 ml IMDM (Life Technologies) with 2% FBS were mixed with 1ml MethoCult H4034 Optimum (STEMCELL Technologies). The mixture was then transferred to 2 wells of ultra-low attachment 24-well plates (Corning). The cells were incubated at 37 °C in 5% CO2 with 100% humidity for 14 days, and then the colonies were counted. Each type of colony was classified according to morphology. Each assay was performed in triplicate.

Shen J., Lyu C., Zhu Y., Feng Z., Zhang S., Hoyle D.L., Ji G., Brodsky R.A., Cheng T, & Wang Z.Z. (2019). Defining early hematopoietic-fated primitive streak specification of human pluripotent stem cells by the orchestrated balance of Wnt, Activin and BMP signaling. Journal of cellular physiology, 234(9), 16136-16147.

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Single Cell Colony Growth Assay

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Single cell-derived colony growth was performed for 14 days in fully cytokine supplemented methylcellulose based medium MethoCult H4034 Optimum (Stem Cell Technologies, Vancouver, BC Canada). Single non-overlapping colonies of different morphologies were picked and DNA extraction was performed using QuickExtract DNA extraction solution on Day 14 of plating (Epicentre, Madison, WI) in two independent experiments. Figure 1B shows pooled data from two independent experiments.

Tothova Z., Krill-Burger J.M., Popova K.D., Landers C.C., Sievers Q.L., Yudovich D., Belizaire R., Aster J.C., Morgan E.A., Tsherniak A, & Ebert B.L. (2017). Multiplex CRISPR-Cas9 Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia. Cell stem cell, 21(4), 547-555.e8.

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