Methocult sf h4236

Manufactured by STEMCELL

Sourced in United States, Canada

MethoCult SF H4236 is a semi-solid culture medium used for colony-forming unit (CFU) assays. It supports the growth and differentiation of human hematopoietic progenitor cells in vitro.

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11 protocols using methocult sf h4236

Clonogenic Assay for BRCA1 Deficiency

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Freshly harvested Lin⁻ murine bone marrow cells were plated in serum-free MethoCult-SF H4236 (StemCell Technologies) supplemented with TET System Approved Fetal Bovine Serum (Takara Bio USA) with and without 10 μg/mL tetracycline hydrochloride in the presence of a threshold concentrations (0.1 U/mL) of recombinant murine IL-3, IL-6, and SCF. Colonies were scored after 5–7 days. BRCA1-deficient and BRCA1-proficient counterpart MDA-MB-436 cells, cultured in RPMI + 10% fetal bovine serum (FBS), were seeded on day 0 in triplicate at 5,000 cells/well. On day 1, the cells were treated with 0, 10, 20, and 50 nM olaparib in the absence or presence of 1 μM D-I03. On day 3, the treatment was repeated with media refreshment. Cells were counted on day 4 using trypan blue exclusion and were immediately plated at a density of 500 cells/well in a six-well plate, in RPMI + 10% FBS. After 2 weeks, cell survival was determined using the clonogenic assay. For this purpose, the colonies were fixed and stained with 0.05% of 10 mg/mL ethidium bromide in 50% ethanol and visualized using an Alphaimager 3400 gel documentation system.

Sullivan-Reed K., Bolton-Gillespie E., Dasgupta Y., Langer S., Siciliano M., Nieborowska-Skorska M., Hanamshet K., Belyaeva E.A., Bernhardy A.J., Lee J., Moore M., Zhao H., Valent P., Matlawska-Wasowska K., Müschen M., Bhatia S., Bhatia R., Johnson N., Wasik M.A., Mazin A.V, & Skorski T. (2018). Simultaneous Targeting of PARP1 and RAD52 Triggers Dual Synthetic Lethality in BRCA-Deficient Tumor Cells. Cell reports, 23(11), 3127-3136.

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IgA1 Depletion Affects Erythropoiesis

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Human CD34+ cord blood cells were cultured on methylcellulose (Methocult SF H4236, stemcell technologies, Vancouver, Canada) with interleukin-3 (IL-3), IL-6, SCF and Epo (0.05 or 1 U/mL) in the presence of sera from IgAN-PE or IgAN patients or healthy volunteers. Depletion of IgA1 was performed as previously described.⁶ Briefly, sera were passed through a Jaccalin column to catch IgA1. We obtained IgA1-depleted serum on one hand, and purified IgA1 after elution of the column on the other hand. BFU-E–derived colonies in the presence of depleted and non-depleted sera were counted at day 14.

Cohen C., Coulon S., Bhukhai K., Neuraz A., Dussiot M., Fouquet G., Stang M.B., Flamant M., Vrtovsnik F., Hummel A., Knebelmann B., Mesnard L., Rondeau E., Maciel T.T., Favale F., Casadevall N., Nguyen-Khoa T., Moutereau S., Legendre C., Benhamou M., Monteiro R.C., Hermine O., El Karoui K, & Moura I.C. (2021). Erythrocytosis associated with IgA nephropathy. EBioMedicine, 75, 103785.

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Colony Formation Assay for CD133+ Cells

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1 × 10³ CD133⁺ cells were plated in triplicates on a 35-mm dish in supplemented MethoCult SF H4236 (Stemcell Technologies Inc.) 2.5 h after the manufacturing process as previously described [28 (link)] in accordance to OECD principles for GLP. After 14 days of incubation at 37 °C and 5% CO₂, adherent and non-adherent colonies were counted.

Skorska A., Müller P., Gaebel R., Große J., Lemcke H., Lux C.A., Bastian M., Hausburg F., Zarniko N., Bubritzki S., Ruch U., Tiedemann G., David R, & Steinhoff G. (2017). GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration. Stem Cell Research & Therapy, 8, 33.

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Expansion and Characterization of CD34+ EPCs

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CD34⁺ cells from the HUCB were expansion-cultured as previously reported (Masuda et al., 2011 (link)). For the EPC colony forming assay, expanded CD34⁺ cells (1000 cells per 35-mm dish) were cultured for 14–21 days in methylcellulose-containing medium (MethoCult SF H4236, StemCell Technologies, Canada) with 20 ng/ml recombinant human stem cell factor (Peprotech, USA), 50 ng/ml vascular endothelial growth factor (VEGF; Peprotech), 20 ng/ml interleukin-3 (Peprotech), 50 ng/ml basic fibroblast growth factor (Peprotech), 50 ng/ml epidermal growth factor (Peprotech), 50 ng/ml insulin-like growth factor-1 (Peprotech), 2 U/ml heparin (Sigma-Aldrich), 1% penicillin-streptomycin (Welgene, Korea), and 30% fetal bovine serum (Thermo Fisher Scientific, USA). The colony forming ability was measured by counting the number of small and large EPC colony forming units (CFU) respectively.

Kim Y.J., Ji S.T., Kim D.Y., Jung S.Y., Kang S., Park J.H., Jang W.B., Yun J., Ha J., Lee D.H, & Kwon S.M. (2018). Long-Term Priming by Three Small Molecules Is a Promising Strategy for Enhancing Late Endothelial Progenitor Cell Bioactivities. Molecules and Cells, 41(6), 582-590.

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Hematopoietic Colony Assay from Tumor Cells

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To observe hematopoietic colony-forming unit (CFU) formation, the cell suspension obtained from tumor tissue was seeded in methylcellulose media: (MethoCult H4230 and MethoCult SF H4236, Stemcell Technologies) according to manufacturer’s protocol. Both media were supplemented with IL-3 (20 ng/mL), IL-6 (20 ng/mL), G-CSF (20 ng/mL), GM-CSF (20 ng/mL), SCF (50 ng/mL) and erythropoietin (3 units/mL). After incubation for 14–16 days at 37 °C with 5 % CO₂, the colonies were characterized and scored according to their morphology on a ZEISS AX10 Inverted Microscope (Zeiss).

Lu I.N., Dobersalske C., Rauschenbach L., Teuber-Hanselmann S., Steinbach A., Ullrich V., Prasad S., Blau T., Kebir S., Siveke J.T., Becker J.C., Sure U., Glas M., Scheffler B, & Cima I. (2021). Tumor-associated hematopoietic stem and progenitor cells positively linked to glioblastoma progression. Nature Communications, 12, 3895.

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Generation and Culture of MRGPRX2+ PBCMCs

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Human PBCMCs were generated as described previously (18 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy donors or rocuronium-hypersensitive patients using Histopaque. Next, CD34⁺ progenitors were isolated from PBMCs (EasySep Human CD34 Selection Kit, Stemcell Technologies) and cultured, for 4-5 weeks, in serum-free methylcellulose-based medium (MethoCult SF H4236, Stemcell Technologies) supplemented with penicillin (100 units/mL, Life Technologies), streptomycin (100 μg/mL, Life Technologies), low-density lipoprotein (LDL, 10 μg/mL, Stemcell Technologies), 2-mercaptoethanol (55 μmol/L, Life Technologies), stem cell factor (SCF, 100 ng/mL, Miltenyi Biotec), interleukin-3 (IL-3, 100 ng/mL, PeproTech) and interleukin-6 (IL-6, 50 ng/mL, Miltenyi Biotec). PBCMCs harbor a MRGPRX2⁺ and a MRGPRX2^- subpopulation (Supplementary Figure 1). Each PBCMC culture yielded 75 ± 5% MRGPRX2⁺ cells (n=15).

Elst J., Maurer M., Sabato V., Faber M.A., Bridts C.H., Mertens C., Van Houdt M., Van Gasse A.L., van der Poorten M.L., De Puysseleyr L.P., Hagendorens M.M., Van Tendeloo V.F., Lion E., Campillo-Davo D, & Ebo D.G. (2021). Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones. Frontiers in Immunology, 12, 668962.

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Clonogenic Potential of Colorectal Cancer Stem Cells

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To assess the clonogenic potential of CRC-SCs, 200 cells were seeded in ultra-low 24-well plate in a final volume of 800 µL of methylcellulose (2 parts of MethoCult SF H4236, Stemcell Technologies, Inc., Vancouver, Canada, and 1 part of complete medium). Cells were incubated at 37 °C, 5% CO₂ for 12 days. Subsequently, colonies of two wells were stained with 0.5 mg/ml MTT for quantification and area analysis, whereas colonies of the two remaining wells were disaggregated and resuspended in a complete fresh medium. Next, following the aforementioned protocol, a second plating of the obtained homogeneous cell suspension was performed. Lastly, pictures were acquired and analyzed with ImageJ Software v.1.52a (Ashland, OR, USA).

Antona A., Leo G., Favero F., Varalda M., Venetucci J., Faletti S., Todaro M., Mazzucco E., Soligo E., Saglietti C., Stassi G., Manfredi M., Pelicci G., Corà D., Valente G, & Capello D. (2023). Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation. Cell Death Discovery, 9, 201.

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Clonogenic Assay for BRCA1 Deficiency

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Endothelial Progenitor Cell Assay

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Cells were placed in an EPC‐colony‐forming assay (CFA) semisolid culture medium, MethoCult SF H4236 (Stem Cell Tech.), supplemented with rhVEGF (50 ng/mL), rhFGF‐2 (50 ng/mL), rhSCF (100 ng/mL), rhIGF‐1 (50 ng/mL), rhIL‐3 (20 ng/mL), rhEGF (50 ng/mL) (PeproTech), 30% FBS (CCB, Nichirei Biosci., Tokyo, Japan), heparin (2 IU/mL) (Ajinomoto, Tokyo, Japan), and an antibiotic cocktail. EPC‐CFA working medium was diluted with 30% FBS‐IMDM, and 1 mL of suspended PbMNCs was seeded with blunt‐end needles (Nipro, Osaka, Japan) attached to 1‐mL syringes into a 35‐mm Primaria culture dish to promote cell adhesion (2 × 10⁵ cells/dish). On day 16, the number of adherent colonies per dish was measured using a gridded scoring dish (Nunc, Thermo Fisher Scientific) using a phase‐contrast light microscope (Eclipse Ti‐U; Nikon, Tokyo, Japan). Based on the maturation stage of colony‐forming cells, EPCs were classified as primitive EPC colony‐forming units (pEPC‐CFUs) or definitive EPC (dEPC)‐CFUs.

Tanaka R., Ito‐Hirano R., Fujimura S., Arita K., Hagiwara H., Mita T., Itoh M., Kawaji H., Ogawa T., Watada H., Masuda H., Asahara T, & Mizuno H. (2021). Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing. Stem Cells Translational Medicine, 10(6), 895-909.

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Isolation and Culture of PBCMC Subpopulations

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Human PBCMCs were cultured as previously described 5, 8 . Briefly, peripheral blood mononuclear cells were isolated from 50 mL of fresh peripheral blood from healthy volunteers, and CD34 + progenitor cells were enriched using the EasySep™ Human CD34 Selection Kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Isolated peripheral blood CD34 + progenitor cells were cultured in a serum-free methylcellulose-based medium (MethoCult™ SF H4236; STEMCELL Technologies) supplemented with penicillin (100 units/mL; Life Technologies, Waltham, MA, USA), streptomycin (100 mg/mL; Life Technologies), low-density lipoprotein (10 mg/mL; STEMCELL Technologies), 2-mercaptoethanol (55 mmol/L; Life Technologies), stem cell factor (100 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), interleukin-3 (100 ng/mL; PeproTech, Rocky Hill, NJ, USA), and interleukin-6 (50 ng/mL; Miltenyi Biotec) for 4-5 weeks.
PBCMCs harbour a MRGPRX2 + and a MRGPRX2 -subpopulation, relevant to our further experiments (Suppl. Figure 1).

Ebo D.G., Elst J., Moonen N., van der Poorten M.M., Van Gasse A.L., Garvey L.H., Bridts C.H., Mertens C., Hagendorens M.M, & Sabato V. (2022). Mast cell activation test: A new asset in the investigation of the chlorhexidine cross-sensitization profile. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 52(11).

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