Methocult h4435 enriched

Manufactured by STEMCELL

Sourced in Canada

MethoCult H4435 Enriched is a methylcellulose-based medium for the colony-forming unit (CFU) assay. It is designed to support the growth and differentiation of human hematopoietic progenitor cells from bone marrow, cord blood, or peripheral blood samples.

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Lab products found in correlation

Smartdish, by STEMCELL (3 mentions) Stemvision human, by STEMCELL (3 mentions) Methocult gf m3434 medium, by STEMCELL (2 mentions) Methocult gf m3434, by STEMCELL (2 mentions) Facsaria 2, by BD (2 mentions) Eclipse ti u, by Nikon (2 mentions) Maxwell 16 cell lev dna purification kit, by Promega (1 mentions) Fetal bovine serum (fbs), by STEMCELL (1 mentions) Alpha mem, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions) Human erythropoietin, by Amgen (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Biorevo bz 900 microscope, by Keyence (1 mentions) Accuprime pfx supermix, by Thermo Fisher Scientific (1 mentions) Ck2 inverted microscope, by Olympus (1 mentions) Cc100 cytokine cocktail, by STEMCELL (1 mentions) Stemspan serum, by STEMCELL (1 mentions) Methocult h4531, by STEMCELL (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions)

Spelling variants of this product

MethoCult (224 citations) MethoCult H4435 (73 citations) H4435 (18 citations) MethoCult Enriched (8 citations) MethoCult H4435 Enriched medium (7 citations) Methocult H4435 Enriched methylcellulose (5 citations) MethoCult H4435 Enriched Media (2 citations)

38 protocols using methocult h4435 enriched

Clonogenic Hematopoietic Progenitor Assay

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For population level assay, cells were sorted using FACS and seeded onto fibronectin coated dishes (Fibronectin Human Protein, Plasma, 4 μg/cm², GIBCO, cat# 33016015) as described in Dege and Sturgeon (2017) , with MethoCult H4435 Enriched (STEMCELL Technologies, cat# 4435). For single-cell level assay, cells were directly sorted into fibronectin coated 96-well plates using FACS. Afterward, a P1000 tip with the tip trimmed to widen its bore was used to gently resuspend each well of the seeded methylcellulose media in a tissue culture hood to ensure optimal growth.
CFU-E erythroid colony and BFU-E erythroid colony were analyzed 8 days after plating. CFU-GM granulocyte-monocyte colony and CFU-GEMM multipotent hematopoietic progenitor colony were analyzed 14 days after plating.

Qiu J., Nordling S., Vasavada H.H., Butcher E.C, & Hirschi K.K. (2020). Retinoic Acid Promotes Endothelial Cell Cycle Early G1 State to Enable Human Hemogenic Endothelial Cell Specification. Cell reports, 33(9), 108465.

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Progenitor Frequency Evaluation of HSPC

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To evaluate the progenitor frequency of HSPCs after being electroporated and cultured for 3 days, 1e4 cells were harvested from each condition and added to MethoCult™ H4435 Enriched (StemCell Technologies) tubes at 250 cells/dish (two MethoCult™ tubes total per test condition). The contents of each tube were then plated into 3 replicate 35 mm dishes. After 15 days of culture at 37 °C and 5% CO₂, the total number of myeloid (CFU-GM), erythroid (BFU-E) and mixed (CFU-GEMM) colonies were enumerated based on morphology. Once counting was complete, photographs were taken of representative colonies from each test condition. The colony number for each test condition was tabulated.
Individual erythroid and myeloid colonies were harvested by plucking single colonies and pipetting them into one well of a 96-well V-bottom plate containing 150 μL of PBS + 2% FBS. Colonies were then pelleted by centrifugation at 739 g for 5 min, supernatant carefully removed and the cell pellets stored at −80 °C. For gDNA extraction, pellets were thawed to room temperature and resuspended in appropriate volume of QuickExtract. Samples were then cycled in a PCR machine at 65 °C for 15 min, 68 °C for 15 min, 98 °C for 10 min. Samples were then frozen at −20 °C. Samples for Next Generation Sequencing (NGS) were prepared and analyzed as described above.

McGaw C., Garrity A.J., Munoz G.Z., Haswell J.R., Sengupta S., Keston-Smith E., Hunnewell P., Ornstein A., Bose M., Wessells Q., Jakimo N., Yan P., Zhang H., Alfonse L.E., Ziblat R., Carte J.M., Lu W.C., Cerchione D., Hilbert B., Sothiselvam S., Yan W.X., Cheng D.R., Scott D.A., DiTommaso T, & Chong S. (2022). Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. Nature Communications, 13, 2833.

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Clonogenic Hematopoietic Progenitor Assay

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Clonogenic Assay of Hematopoietic Cells

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Single CD34+ cell-derived CFUs were grown for 14 days in MethoCult™ H4435 Enriched (STEMCELL Technologies). For infusion product CFUs, CD34+ cells electroporated with Cas9 RNP were incubated for 24 hours at 32°C, then plated with 1.1ml of MethoCult H4435 in 35-mm CFU dishes at a density of 2000 cells/dish. For ZL33’s 4.5m BM CFUs, 20 ml of BM aspirate was collected, and CD34+ cells were immunoselected as previously described (Donahue et al., 2005 ; Wu et al., 2014 (link)). BM-derived CD34+ cells were plated with 1.1ml of MethoCult H4435 in 35-mm CFU dishes at a density of 2000 cells/dish. The remaining cells were used for targeted deep sequencing. Single CFU colonies were picked, and DNA was extracted with the Maxwell® 16 Cell LEV DNA Purification Kit (Promega, AS1140) on day 14 of plating.

Kim M.Y., Yu K.R., Kenderian S.S., Ruella M., Chen S., Shin T.H., Aljanahi A.A., Schreeder D., Klichinsky M., Shestova O., Kozlowski M.S., Cummins K.D., Shan X., Shestov M., Bagg A., Morrissette J.J., Sekhri P., Lazzarotto C.R., Calvo K.R., Kuhns D.B., Donahue R.E., Behbehani G.K., Tsai S.Q., Dunbar C.E, & Gill S. (2018). Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell, 173(6), 1439-1453.e19.

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Single-cell HSPC Colony Assay

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Single HSPCs (7AAD⁻, Lin⁻, CD34⁺) were sorted into 96-well plates with 50 μl of MethoCult H4435 Enriched (STEMCELL Technologies). The mean fluorescence intensities of CD34, CD38, CD90, CD45RA and CD123 were also recorded for each individual HSPC sorted using the index sorting feature. Colony output was assessed on day 14 under direct light microscopy, and individual colonies were picked. All colonies were flash-frozen and stored at −80^oC for subsequent whole-genome amplification. Index-sorting analysis was performed by in-house bioinformatics pipelines using R studio v3.6.3.

Sousos N., Ní Leathlobhair M., Simoglou Karali C., Louka E., Bienz N., Royston D., Clark S.A., Hamblin A., Howard K., Mathews V., George B., Roy A., Psaila B., Wedge D.C, & Mead A.J. (2022). In utero origin of myelofibrosis presenting in adult monozygotic twins. Nature Medicine, 28(6), 1207-1211.

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In vitro Erythropoiesis and VCN Determination

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CD34+ cell were seeded at 0.5 – 1 × 10⁶/mL in a 6 well plate containing Alpha MEM (Sigma), FBS (Stem Cell Technologies), Glutamine (Gibco), BSA (Sigma), human erythropoietin (Amgen), β-mercaptoethanol (Gibco), dexamethasone (American Regent Laboratories), holo-transferrin (American Regent Laboratories) and human recombinant SCF (Sigma). Cells were counted and fed from day 4 to 10 with the same EC differentiation medium. This differentiation process was used for in vitro modeling of erythropoiesis and VCN determination. For CD34+ cells pre- and post-transduction, CD34+ cells were plated at 500 cells per plate in MethoCultH4435 Enriched (StemCell Technology). BFU-E, CFU-M, CFU-G and CFU-GM were distinguished and counted after 14 – 16 days as per as per manufacturers’ directions.

Boulad F., Maggio A., Wang X., Moi P., Acuto S., Kogel F., Takpradit C., Prockop S., Mansilla-Soto J., Cabriolu A., Odak A., Qu J., Thummar K., Du F., Shen L., Raso S., Barone R., Di Maggio R., Pitrolo L., Giambona A., Mingoia M., Everett J.K., Hokama P., Roche A.M., Cantu V.A., Adhikari H., Reddy S., Bouhassira E., Mohandas N., Bushman F.D., Rivière I, & Sadelain M. (2022). A phase 1 trial of lentiviral globin gene therapy with reduced intensity conditioning in adults with transfusion dependent β-thalassemia. Nature medicine, 28(1), 63-70.

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Hematopoietic Progenitor Assessment

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Hematopoietic progenitors were assessed after treatment with ABT-199 (1 μM) and DMSO (0.001%) for 72 h in cytokine-supplemented, serum-free culture. 1 × 10⁴ BMMNC were plated in duplicates in methylcellulose medium supplemented with an optimal cytokine mix according to the manufacturer’s protocols (MethoCult H4435 enriched; StemCell Technologies). Numbers of erythroid progenitor colonies (Burst-forming units-erythroid or colony-forming units for the granulocytic-macrophagic lineage, and multi-potential granulocytic-erythroid-macrophagic-megakaryocytic lineage) were assessed after 14 days. Transmitted light photographs were obtained on a Keyence BIOREVO BZ-900 microscope.

Reidel V., Kauschinger J., Hauch R.T., Müller-Thomas C., Nadarajah N., Burgkart R., Schmidt B., Hempel D., Jacob A., Slotta-Huspenina J., Höckendorf U., Peschel C., Kern W., Haferlach T., Götze K.S., Jilg S, & Jost P.J. (2018). Selective inhibition of BCL-2 is a promising target in patients with high-risk myelodysplastic syndromes and adverse mutational profile. Oncotarget, 9(25), 17270-17281.

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Murine and Human HSC Colony Formation

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Murine or human HSCs were resuspended in complete MethoCult GF M3434 medium or MethoCult H4435 Enriched (STEMCELL Technologies), grown at 37°C in a 5% CO₂ chamber, was monitored until colonies were visible (7 and 21 days, respectively). Number of colonies and colony type were quantified manually.

Lengefeld J., Cheng C.W., Maretich P., Blair M., Hagen H., McReynolds M.R., Sullivan E., Majors K., Roberts C., Kang J.H., Steiner J.D., Miettinen T.P., Manalis S.R., Antebi A., Morrison S.J., Lees J.A., Boyer L.A., Yilmaz Ö.H, & Amon A. (2021). Cell size is a determinant of stem cell potential during aging. Science Advances, 7(46), eabk0271.

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Colony Forming Assay and Genotyping

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One day after electroporation, 1000 CD34+ cells were plated in 1.1ml of methylcellulose (MethoCult H4435 Enriched, Stem Cell Technologies) on 6 well plates in duplicate and cultured for two weeks, after which colonies were counted and scored. Individual colonies were picked and heat lysed in 40ul of lysis buffer containing 50mM NaOH and 0.2mM EDTA. Samples were heated to 95°C for 20 minutes then cooled down, after which 1ul of 1M TrisCl was added. 2ul of reaction was used for PCR with AccuPrime Pfx SuperMix (Invitrogen, 12344-040) as per manufacturer’s instructions. Alternatively, MethoCult wells were solubilized with RPMI media overnight and flow cytometry was performed on single-cell suspensions.

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Hematopoietic Colony Forming Assay

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Methocult H4435 Enriched (Stemcell Technologies) was used for the colony forming unit (CFU) assays where 5000 or 20 000 cells were plated/petri dish and incubated for 14 days according to the manufacturer's protocol.

Nilsson T., Waraky A., Östlund A., Li S., Staffas A., Asp J., Fogelstrand L., Abrahamsson J, & Palmqvist L. (2022). An induced pluripotent stem cell t(7;12)(q36;p13) acute myeloid leukemia model shows high expression of MNX1 and a block in differentiation of the erythroid and megakaryocytic lineages. International Journal of Cancer, 151(5), 770-782.

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