Methocult gf 3434

Manufactured by STEMCELL

Sourced in Canada

MethoCult GF 3434 is a methylcellulose-based medium for the detection and enumeration of human hematopoietic progenitor cells in in vitro colony-forming unit (CFU) assays. It contains recombinant human growth factors, including stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, and erythropoietin.

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Lab products found in correlation

Ack lysing buffer, by Lonza (2 mentions) Coulter counter, by Beckman Coulter (2 mentions) Methocult m3630, by STEMCELL (1 mentions) Trypan blue, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hanks balanced salt solution with ca2 and mg2, by Thermo Fisher Scientific (1 mentions) Rat anti mouse cd16 32 antibody, by BD (1 mentions) 7 aad, by BD (1 mentions) Pe cy5 conjugated lineage cocktail, by Thermo Fisher Scientific (1 mentions) Pe conjugated anti mouse sca1 and apc conjugated anti mouse c kit, by Thermo Fisher Scientific (1 mentions) Facscanto, by BD (1 mentions)

Spelling variants of this product

MethoCult (224 citations) MethoCult 3434 (23 citations) MethoCult GF (18 citations) MethoCult™ GF 3434 Media (2 citations) MethoCult GF 3434 StemSpan media (2 citations)

11 protocols using methocult gf 3434

Hematopoietic Progenitor Quantification

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Colony-forming cell (CFC) or colony-forming unit (CFU) assays were performed by plating blood in methylcellulose medium for mouse cells supplemented with recombinant cytokines (MethoCult GF 3434; Stem Cell Technologies, Vancouver, BC Canada) according to standard techniques [15 (link)]. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semi-solid media in response to cytokine stimulation. The colonies formed can be enumerated and characterized according to their unique morphology. This aims to study differences in the percentage of leukemia CFU/total APL number plated.

Macanas-Pirard P., Quezada T., Navarrete L., Broekhuizen R., Leisewitz A., Nervi B, & Ramírez P.A. (2017). The CCL2/CCR2 Axis Affects Transmigration and Proliferation but Not Resistance to Chemotherapy of Acute Myeloid Leukemia Cells. PLoS ONE, 12(1), e0168888.

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Quantification of Myeloid and Pre-B Cells

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Bone marrow cells (2.5 × 10³) were seeded into methylcellulose-containing media to quantify the absolute number of myeloid progenitors (MethoCult GF3434) and pre–B cells (MethoCult M3630; both, Stem Cell Technologies).
Detailed descriptions of RNA-sequencing, single-cell RNA-sequencing, and data analysis are presented in the supplemental Methods.

Kurtova A.V., Heinlein M., Haas S., Velten L., Dijkgraaf G.J., Storm E.E., Kljavin N.M., Boumahdi S., Himmels P., Herault A., Mancini A., Koeppen H., Dail M., Yan Q., Zhang J., Koch U., Radtke F., Modrusan Z., Metcalfe C., Piskol R, & de Sauvage F.J. (2022). Disruption of stem cell niche–confined R-spondin 3 expression leads to impaired hematopoiesis. Blood Advances, 7(4), 491-507.

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Definitive Hematopoietic Colonies from Fetal Liver

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Individual fetal livers were isolated from E14.5 PDE2A^+/+ and PDE2A^−/− embryos and mechanically dissociated into single cells suspension in CMF buffer. After centrifugation at 1200 rpm for 5 min at 4 °C, cells were resuspended in IMDM, 2% FBS (Gibco BRL), counted in Trypan Blue (Sigma-Aldrich) and plated in triplicate at 20 × 10³ cells/mL, in MethoCult GF 3434 (Stemcell Technology, Vancouver, Canada). Embryonic definitive hematopoietic colonies (BFU-E, CFU-GM, CFU-G CFU-M and CFU-GEMM) were scored after 7 days in a fully humidified incubator at 37 °C with 5% CO₂. Colonies were counted blindly respect to genotype.

Barbagallo F., Rotilio V., Assenza M.R., Aguanno S., Orsini T., Putti S., Isidori A.M., Lenzi A., Naro F., De Angelis L, & Pellegrini M. (2020). PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis. International Journal of Molecular Sciences, 21(8), 2902.

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Isolation and Culture of Murine Hematopoietic Stem Cells

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Whole bone marrow cells were isolated from the femurs and tibias of mice (n = 2–3 per genotype) by grinding the bones in hematopoietic stem cell buffer [Hank’s Balanced Salt Solution with Ca²⁺ and Mg²⁺ (Gibco^®, Grand Island, NY), 5% fetal bovine serum and 2 mM EDTA]. ACK lysing buffer (Lonza, Morristown, NJ) was utilized to deplete red blood cells. After red blood cell lysis, the cells were counted using a Coulter counter (Beckman Coulter^® Inc., Fullerton, CA) and plated in duplicate in MethoCult™ GF 3434 (STEMCELL™ Technologies Inc., Vancouver, Canada). Cells were irradiated 1 h after plating and colonies were counted 7 days later by a single blinded observer.

Castle K.D., Daniel A.R., Moding E.J., Luo L., Lee C.L, & Kirsch D.G. (2018). Mice Lacking RIP3 Kinase are not Protected from Acute Radiation Syndrome. Radiation research, 189(6), 627-633.

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Isolation and Analysis of Mouse Hematopoietic Stem Cells

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Whole bone marrow cells were isolated from femurs and tibias by grinding the bones in hematopoietic stem cell (HSC) buffer [Hanks’ balanced salt solution (HBSS) with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA]. Red blood cells (RBCs) were lysed using ACK lysing buffer (Lonza, Basel, Switzerland). The total cell number was counted with a Coulter counter (Beckman Coulter Inc., Brea, CA). Three million cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail (anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies), PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (eBioscience). Dead cells were excluded by staining with 7-AAD (BD Pharmigen). Data were collected from 1 million single cells by FACSCanto (BD Biosciences) and analyzed by FlowJo software.
For colony-forming cell (CFC) assays, 2 × 10⁴ RBC-depleted whole bone marrow cells were plated in MethoCult GF 3434 (StemCell Technologies) and colonies were counted 7 days later.

Lee C.L., Lento W.E., Castle K.D., Chao N.J, & Kirsch D.G. (2014). Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in Mice. Radiation research, 181(5), 445-451.

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Colony Forming Assay for Hematopoietic Cells

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The colony forming assay was performed with growth factor‐supplemented methylcellulose (MethoCult GF 3434; Stemcell Technologies). A total of 1 × 10⁴ Lin⁻ BM cells were seeded into methylcellulose on two 3.5 cm dishes. After 10 days of culture at 37°C, 5% CO₂, and more than 95% humidity, the colonies formed were counted under the microscope.

Hausinger R., Hackl M., Jardon Alvarez A., Kehr M., Romero Marquez S., Hettler F., Kehr C., Grziwok S., Schreck C., Peschel C., Istvánffy R, & Oostendorp R.A. (2021). Cathepsin K maintains the compartment of bone marrow T lymphocytes in vivo. Immunity, Inflammation and Disease, 9(2), 521-532.

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Quantifying Bone Marrow Progenitors

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Whole bone marrow cells were harvested following the procedure described previously^{3 (link)}. 2 × 10⁴ whole bone marrow cells were plated in MethoCult GF 3434 (Stem cell technologies). Whole bone marrow cells were irradiated about 1 h after plating and the number of colonies was counted 7 days later. A colony is defined as clusters with a minimal of 30 cells. Colonies were counted without knowledge of the genotype or treatment by two observers (SH and C-LL).

Hasapis S., Caraballo I., Sears T.J., Brock K.D., Cart J.B., Moding E.J, & Lee C.L. (2023). Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice. Scientific Reports, 13, 12916.

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Bone Marrow Colony-Forming Assay

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Bone marrow mononuclear cells (BMMC) were isolated from Jak2^w/w (n = 3) and Jak2^w/VF (n = 3) mice by Ficoll‐gradient centrifugation. In total, 1 × 10⁵ cells were plated onto methocult GF3434 (StemCell Technologies Inc, Vancouver, Canada) in the presence or not of ruxolitinib (300 nmol/L) and/or obatoclax (250 nmol/L). Colonies were counted under an inverted microscope after 10 d of culture and classified according to their morphology into granulocyte‐monocyte colony‐forming unit (CFU‐GM), granulocyte colony‐forming unit (CFU‐G), monocyte colony‐forming unit (CFU‐M), erythroid colony‐forming unit (BFU‐E) or colony‐forming unit of granulocytes, erythrocytes, monocytes and megakaryocytes (CFU‐GEMM).

Takei H., Coelho‐Silva J.L., Tavares Leal C., Queiroz Arantes Rocha A., Mantello Bianco T., Welner R.S., Mishima Y., Kobayashi I.S., Mullally A., Lima K., Machado‐Neto J.A., Kobayashi S.S, & Lobo de Figueiredo‐Pontes L. (2021). Suppression of multiple anti‐apoptotic BCL2 family proteins recapitulates the effects of JAK2 inhibitors in JAK2V617F driven myeloproliferative neoplasms. Cancer Science, 113(2), 597-608.

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Evaluating Hematopoietic Stem Cell Potential

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Following bone marrow transduction, GFP positive cells were isolated by FACS and plated in methylcellulose medium (Methocult GF 3434, Stem Cell Technologies). Colony formation was evaluated after 10–14 days; clusters containing more than 30 cells were scored as a colony. Secondary colony formation was tested by harvesting entire primary colony cultures, washing the cells two times with PBS, and plating 10,000 cells in methylcellulose a second time. Secondary colonies were enumerated 12–14 days following secondary plating. Cells/colony were calculated by dividing the total number of cells obtained per dish by the total number of colonies in that dish. We confirmed using fluorescent microscopy that all colonies continued to maintain transgene expression.

Ichim C.V., Dervovic D.D., Chan L.S., Robertson C.J., Chesney A., Reis M.D, & Wells R.A. (2018). The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo. Biomarker Research, 6, 36.

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Quantifying Hematopoietic Stem Cell Activity

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For in vitro colony formation assays, RBC-depleted WBM cells were isolated and counted following the same procedure described above. WBM cells were plated in MethoCult GF 3434 (Stem cell technologies) and colonies were counted 7 days later. Experiments to assess colony forming unit-spleen 12 (CFU-S₁₂) was performed following the protocol described previously⁵⁸. RBC-depleted WBM cells were isolated and counted following the same procedure described above. WBM cells were transplanted into C57BL/6 mice irradiated with 9.5 Gy TBI. Spleens were harvested 12 days after irradiation and preserved in Bouin’s solution for counting the colonies. Colonies were counted without knowledge of the genotype or treatment by a single observer (CLL).

Lee C.L., Castle K.D., Moding E.J., Blum J.M., Williams N., Luo L., Ma Y., Borst L.B., Kim Y, & Kirsch D.G. (2015). Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma. Nature Communications, 6, 8477.

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