Methocult m3334

Manufactured by STEMCELL

Sourced in Canada

MethoCult M3334 is a methylcellulose-based medium used for the clonogenic assay of mouse hematopoietic progenitor cells. It supports the growth and differentiation of granulocyte, erythroid, macrophage, and megakaryocyte colonies from mouse bone marrow, spleen, and fetal liver cell samples.

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17 protocols using methocult m3334

Enumeration of Erythroid Progenitor Cells

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BM cells and splenocytes were collected and treated with hypotonic buffer as previously described^{19 (link),40 (link)}, plated at different densities in p12-wells and incubated at 37 °C in an atmosphere containing 5% CO₂. For the quantification of BFU-E, methylcellulose-based semi-solid culture medium MethoCult™ SF M3436 (Stem Cell Technologies) was used, following the manufacturer’s instructions. After 9 days of incubation colonies were counted in an inverted microscope at a 10× magnification. For detection of CFU-E, the methylcellulose-based medium MethoCult™ M3334 (Stem Cell Technologies) was used, following the manufacturer’s instructions. After 48 h of incubation, CFU-E were counted in an inverted microscope at 20× magnification.

Sánchez Á., Orizaola M.C., Rodríguez-Muñoz D., Aranda A., Castrillo A, & Alemany S. (2020). Stress erythropoiesis in atherogenic mice. Scientific Reports, 10, 18469.

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Mouse Erythroid Progenitor Assay

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Mouse CFU-E assays was performed according to manufacturer’s instructions (STEMCELL Technologies Technical Manual for Mouse Colony-Forming Unit Assays using MethoCult version 3.4.0). Bone marrow cells (4 × 10⁵) in IMDM with 2% FBS were transferred into 4 mL MethoCult M3334 methylcellulose-based medium containing EPO (STEMCELL Technologies). Samples were vortexed thoroughly and transferred in triplicate (1 × 10⁵ cells) to treated 35-mm culture dishes (STEMCELL Technologies). CFU-E colonies were enumerated after 72 h using an inverted microscope. CFU-E colony counts are reported as number of colonies/10⁶ bone marrow cells.

Zhou X., Medina S., Bolt A.M., Zhang H., Wan G., Xu H., Lauer F.T., Wang S.C., Burchiel S.W, & Liu K.J. (2020). Inhibition of red blood cell development by arsenic-induced disruption of GATA-1. Scientific Reports, 10, 19055.

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Quantifying Hematopoietic Progenitor Colonies

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To detect burst-forming units-erythroid (BFU-E) colonies, 5 X 10⁴ bone marrow cells were seeded in duplicates in semisolid medium (Methocult M3434; STEMCELL Technologies, Vancouver, BC). To detect colony forming units-erythroid (CFU-E) colonies, 8 X 10⁴ bone marrow cells were seeded in duplicates in semisolid medium (Methocult M3334; STEMCELL Technologies, Vancouver, BC). To detect colony- forming units-megakaryocytes (CFU-Mk) colonies, 1 X 10⁵ bone marrow cells were seeded in duplicates in semisolid medium (Methocult-c, 04974; STEMCELL Technologies, Vancouver, BC) supplemented with 10 ng/ml Interleukin (IL)-3, 20 ng/ml Interleukin (IL)-6, 50 ng/ml thrombopoietin (TPO) (STEMCELL Technologies, Vancouver, BC). To analyze the colonies of multi-potential progenitor cells (CFU-GEMM) and granulocyte/macrophage progenitor (CFU-GM), 4 x 10⁴ bone marrow cells were mixed with semisolid medium (Methocult GF M3434; STEMCELL Technologies, Vancouver, BC) by vortexing. Bone marrow cells (4 x 10⁴ cells) from WT mice (n = 4) and Thra1^PV/+ mice (n = 4) were seeded in 6 wells plate (DENVILLE, SCIENTIFIC INC., quadruplicates) which was cultured in 5% CO₂ humidified incubator at 37°C. The numbers of colonies were counted under inverted microscope (Primo Vert, Ziess) by morphologic criteria 8 days after plating.

Park S., Han C.R., Park J.W., Zhao L., Zhu X., Willingham M., Bodine D.M, & Cheng S.Y. (2017). Defective erythropoiesis caused by mutations of the thyroid hormone receptor α gene. PLoS Genetics, 13(9), e1006991.

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Hematopoietic Progenitor Cell Assay

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CFU-E and BFU-E were analyzed using BM cells (5×10⁵ cells/dish), according to the manufacturer’s protocol (StemCell Technologies, Vancouver, Canada). Methocult M3334 (StemCell Technologies) was used for CFU-E and BFU-E colonies determined after 2 days and 9 days of culture, respectively.

Kanematsu T., Suzuki N., Tamura S., Suzuki A., Ishikawa Y., Katsumi A., Kiyoi H., Saito H., Kunishima S., Kojima T, & Matsushita T. (2021). Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice. Nagoya Journal of Medical Science, 83(1), 75-86.

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Hematopoietic Colony Forming Assays

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For detection of CFU-E colonies, aliquots containing 2 × 10⁵ BM cells resuspended in 0.7 mL of erythropoietin-containing methylcellulose culture (MethoCult M3334; StemCell Technologies, Vancouver, Canada) were plated into 12-well plates according to the manufacturer’s protocol, and CFU-E colonies were counted after 2 days of culture. To detect BFU-E, CFU-GM, CFU-M, CFU-G and mixed CFU colonies, aliquots containing 200 CMPs or MEPs resuspended in 0.7 mL of multiple cytokine-containing methylcellulose culture (MethoCult M3434; StemCell Technologies) were plated into 12-well plates, and the colonies were counted after 10 days of culture17 (link)25 (link).

Lin K.R., Yang-Yen H.F., Lien H.W., Liao W.H., Huang C.J., Lin L.I., Li C.L, & Yen J.J. (2016). Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice. Scientific Reports, 6, 31444.

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Quantifying Murine and Human Hematopoietic Progenitors

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BM cells isolated from Fbxo22^+/+ and Fbxo22^−/− or Scl-Cre⁻;Fbxo22^fl/fl and Scl-Cre⁺;Fbxo22^fl/fl mice were plated into 12-well plates containing MethoCult M3434 (StemCell Technologies) for colony-forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) or MethoCult M3534 (StemCell Technologies) for colony-forming unit-granulocyte and macrophage (CFU-GM) colony formation analysis at 2.5 × 10⁴ cells/well, MethoCult M3334 (StemCell Technologies) for colony-forming unit-erythrocyte (CFU-E) or MethoCult M3630 (StemCell Technologies) for colony-forming unit-pre-B lymphocyte (CFU-Pre-B) colony formation analysis at 2.5 × 10⁵ cells/well according to the manufacturer’s protocols. GFP⁺ or LGMP cells sorted from primary AML recipients were plated into 12-well plates containing complete MethoCult M3534 medium at indicated numbers according to the manufacturer’s instructions. For the colony-forming assay of THP-1, FBXO22-knockdown THP-1 cells or control cells were plated into 12-well plates containing complete MethoCult H4436 (StemCell Technologies) at 500 cells/well. For the colony-forming assay of AML patients’ mononuclear cells (MNCs), FBXO22-knockdown or overexpressed cells with control cells were plated into 24-well plates containing MethoCult H4436 at 40,000 cells/well. Colonies were imaged and counted 7–10 days after plating.

Zhu X.N., Wei Y.S., Yang Q., Liu H.R., Zhi Z., Zhu D., Xia L., Hong D.L., Yu Y, & Chen G.Q. (2023). FBXO22 promotes leukemogenesis by targeting BACH1 in MLL-rearranged acute myeloid leukemia. Journal of Hematology & Oncology, 16, 9.

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Erythroid Colony Formation Assay

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BMCs treated with 1, 10, or 100 ng/ml α-toxin for 24 hours in a humidified atmosphere of 95% air with 5% CO₂ at 37 °C were washed and suspended in IMDM containing 10% FBS. The cells were then seeded in MethoCult M3334 (StemCell Technologies) and incubated for 2–10 days in a humidified atmosphere of 95% air with 5% CO₂ at 37 °C in accordance with the manufacturer’s protocol. Subsequently, the number of colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) colonies were counted using a microscope.

Takagishi T., Takehara M., Seike S., Miyamoto K., Kobayashi K, & Nagahama M. (2017). Clostridium perfringens α-toxin impairs erythropoiesis by inhibition of erythroid differentiation. Scientific Reports, 7, 5217.

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Clonogenic Potential of Lung Cancer Spheroids

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Clonogenic growth as spheroids was assessed for H727 and H720 for both parental and third-generation spheroids. Cells were seeded in 1% methylcellulose-based medium (MethoCult™ M3334, STEMCELL, Vancouver, BC, Canada) supplemented with RPMI-1640, 10% FBS and 1% antibiotics (100 μg/ml streptomycin and 100 IU/ml penicillin), cultured in 35 mm tissue culture dishes (Nalgene Nunc International, Rochester, NY, USA) and cultures incubated at 37 °C and 5% CO2. This culture step was repeated two more times and the number of colonies produced by the parental and third-generation spheroids were counted after 7 days using a grading dish on a phase contrast microscope (× 10). The degree of clonogenicity was assessed as the average number of colonies per dish for each cell line and the parental and third-generation spheroids.

Bayat Mokhtari R., Baluch N., Morgatskaya E., Kumar S., Sparaneo A., Muscarella L.A., Zhao S., Cheng H.L., Das B, & Yeger H. (2019). Human bronchial carcinoid tumor initiating cells are targeted by the combination of acetazolamide and sulforaphane. BMC Cancer, 19, 864.

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Quantification of Erythroid Progenitor Cells

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Single-cell suspensions of bone marrow were prepared by flushing the cells out from the femur and tibia using a syringe and a 21-gauge needle. After harvesting under sterile conditions, the spleens were passed through a wire mash and monodispersed in Dulbecco’s modified Eagle’s medium (DMEM-HPA, Capricorn Scientific, Germany) supplemented with 5% fetal calf serum (FBS, Biowes South America No. S1810). Then, 2 × 10⁵/mL bone marrow cells and 4 × 10⁵/mL splenocytes were separately plated in methylcellulose media (StemCell Technologies, Vancouver, BC, Canada) containing either 3 U/mL EPO (MethoCult M3334) or 3 U/mL EPO supplemented with 50 ng/mL SCF, 10 ng/mL IL-3, and 10 ng/mL IL-6 (MethoCult GF M3434). The cells were plated in duplicate in 35 mm tissue culture dishes (Sarstedt, Germany) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. According to the manufacturer’s instructions, burst-forming units-erythroid (BFU-E) were enumerated following the incubation period of 7 days in MethoCult GF M3434 medium, whereas colony-forming unit-erythroid (CFU-E)-derived colonies were scored after 2 days of culture in MethoCult M3334 using an inverted microscope.

Momčilović S., Bogdanović A., Milošević M.S., Mojsilović S., Marković D.C., Kočović D.M, & Vignjević Petrinović S. (2023). Macrophages Provide Essential Support for Erythropoiesis, and Extracellular ATP Contributes to a Erythropoiesis-Supportive Microenvironment during Repeated Psychological Stress. International Journal of Molecular Sciences, 24(14), 11373.

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Hematopoietic Stem Cell Enumeration

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Cell concentration was determined using a hemocytometer (Hausser
Scientific) after staining with Trypan Blue. Two hundred cells were mixed with 3
ml methylcellulose-based medium (Stemcell, MethoCult™ M3334), and plated
in a 35 mm culture dish. Each experimental point contained two duplicate culture
dishes plus one dish filled with water that were held in one 10 cm plate, and
cultured at 37°C with 5% carbon dioxide for five days before
examination under a microscope.

Shi J., Yuan B., Hu W, & Lodish H. (2016). JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating Stat1 and other non-erythroid signaling pathways. Experimental hematology, 44(11), 1044-1058.e5.

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