Methocult sf h4636

Manufactured by STEMCELL

Sourced in Canada, United Kingdom

MethoCult SF H4636 is a methylcellulose-based medium designed for the in vitro culture of human hematopoietic progenitor cells. It contains the necessary components to support the growth and differentiation of various blood cell types, including erythroid, myeloid, and megakaryocytic lineages.

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Lab products found in correlation

Accutase, by STEMCELL (2 mentions) Puromycin dihydrochloride, by Beyotime (1 mentions) Anti cd44 apc, by Thermo Fisher Scientific (1 mentions) Arsenic trioxide, by Merck Group (1 mentions) Igepal co 720, by Merck Group (1 mentions) Anti cd24 fitc, by Thermo Fisher Scientific (1 mentions) Chitosan, by Maclin Biochemical Technology (1 mentions) Coumarin 6, by Merck Group (1 mentions) Apc ef780 conjugated anti human cd31, by Thermo Fisher Scientific (1 mentions) Pe conjugated anti human cd144, by BioLegend (1 mentions) Percp ef710 conjugated anti human cd43, by Thermo Fisher Scientific (1 mentions) Flt3l, by STEMCELL (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Stemspan, by STEMCELL (1 mentions) Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions) Cd34 microbead kit ultrapure human, by Miltenyi Biotec (1 mentions) Cd34 microbead kit ultrapure, by Miltenyi Biotec (1 mentions)

7 protocols using methocult sf h4636

Comprehensive Nanomedicine Protocol Synthesis

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Arsenic trioxide, Igepal CO-720, coumarin 6 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Manganous acetate and chitosan were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). MPEG_5K-PLGA_20K and mPEG_5K-PLA_20K were purchased from Yare Biotech, Inc. (Shanghai, China). Mal-PEG_5K-PLGA_20K was synthesized in Hunan HuaTeng Co., Ltd. (Changsha, China). DSS6 (Ac-CDSSDSSDSSDSSDSSDSS) peptide was synthesized in Top-peptide Co., Ltd. (Shanghai, China). Hydroxyapatite (HA) was obtained from Emperor Nano Material Co., Ltd. (Nanjing, China). 1,1ʹ-Dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindotricarbocyanine iodide (DiR) was purchased from Meilun Biotech Co., Ltd. (Dalian, China). CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Puromycin dihydrochloride was purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Anti-CD44-APC and anti-CD24-FITC were purchased from eBioscience (San Diego, CA, USA). MethoCult™ SF H4636 was purchased from STEMCELL Technologies Inc. (Vancouver, Canada).

Liu C., Hu A., Chen H., Liang J., Gu M., Xiong Y, & Mu C.F. (2021). The osteogenic niche-targeted arsenic nanoparticles prevent colonization of disseminated breast tumor cells in the bone. Acta Pharmaceutica Sinica. B, 12(1), 364-377.

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Endothelial Progenitor Cell Isolation

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After 6 days of culture, EBs were collected, disaggregated, and stained with APC-eF780 conjugated anti-human CD31 (Thermo Fisher Scientific), PE-conjugated anti-human CD144 (BioLegend), PerCP-eF710-conjugated anti-human CD43 (Thermo Fisher Scientific) and Hoechst 33258 (Thermo Fisher Scientific). Live CD31⁺CD144⁺CD43^- cells were sorted and replated for in StemSpan (STEMCELL Technologies) supplemented with 5ng/ml VEGF, 5ng bFGF, 25ng/ml IGF1, 25ng/ml IGF2, 50ng/ml SCF, 50ng/ml TPO, 5ng/ml IL-11, 20ng/ml Flt3-L (all cytokines from PeproTech). Colony forming units (CFU) were performed using MethoCult™ SF H4636 (Stem Cell Technologies).

Menegatti S., Potts B., Paredes R., Garcia-Alegria E., Baker S.M, & Kouskoff V. (2023). CD82 expression marks the endothelium to hematopoietic transition at the onset of blood specification in human. iScience, 26(9), 107583.

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Hematopoietic Stem Cell Differentiation Assay

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HSPCs (1000) collected from 3D hydrogels at day 10 were cultured in MethoCult™ SF H4636 (Stemcell Technologies, Inc) for 14 days as per manufacturer’s protocol. Burst forming unit-erythroid (BFU-E), colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) and colony-forming unit for granulocytes and macrophages (CFU-GM) were identified using EVOS M5000 imaging system (ThermoFisher Scientific). Cells collected from Methocult on day 14 were subjected to immunophenotype analysis using flow cytometry.

Sidhu I., Barwe S.P., Kiick K.L., Kolb E.A, & Gopalakrishnapillai A. (2021). A 3-D Hydrogel Based System for Hematopoietic Differentiation and its use in Modeling Down Syndrome Associated Transient Myeloproliferative Disorder. Biomaterials science, 9(18), 6266-6281.

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Colony-forming unit assay for hematopoietic cells

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MethoCult colony-forming unit (CFU) assays were performed with MethoCult SF H4636 (StemCell Technologies, Cambridge, UK) according to the manufacturer’s protocol. Briefly, on day 9, cells from EBs were dissociated with Accutase and CD34⁺ cells were MACS-purified with the human CD34 UltraPure MicroBead Kit (Miltenyi Biotech, Paris, France). After washing, 10,000 cells were resuspended in 300 mL EB medium, gently mixed with 3 mL MethoCult, and plated onto 2.35 mm dishes. Cord blood CD34⁺ cells (2C-101, Lonza, Colmar, France) were thawed according to the manufacturer’s instructions, washed in DPBS, and 2000 cells were seeded as described above. The cells were incubated at 37 °C and 5% CO₂ for 14 days. The total colony numbers were quantified in duplicate plates using an inverted light microscope, and the average number of CFUs per plate was determined. The MethoCult was then rehydrated to recover the cells. The cells were washed 3x with DPBS and then analyzed by flow cytometry for the expressions of CD15, CD14, and CD235a on the different cell types.

Flippe L., Gaignerie A., Sérazin C., Baron O., Saulquin X., Anegon I., David L, & Guillonneau C. (2022). Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells. Cells, 11(24), 4046.

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Clonogenic Assay of Embryoid Bodies

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Cells from day 21 heX-embryoids were dissociated into single-cell solution by application of Accutase (StemCell Technologies) for 30 min. CFU assays were performed using methylcellulose containing media (MethoCult SF H4636, StemCell Technologies) according to the manufacturer’s instructions. After 14 days in culture, colonies were analysed by image acquisition using light microscopy.

Hislop J., Song Q., Keshavarz F. K., Alavi A., Schoenberger R., LeGraw R., Velazquez J.J., Mokhtari T., Taheri M.N., Rytel M., Chuva de Sousa Lopes S.M., Watkins S., Stolz D., Kiani S., Sozen B., Bar-Joseph Z, & Ebrahimkhani M.R. (2023). Modelling post-implantation human development to yolk sac blood emergence. Nature, 626(7998), 367-376.

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CD34+ Cell Isolation and CFU Assay

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Cells were dissociated into single cell solution using accutase (StemCell Technologies). CD34⁺ cells were purified from a portion of the cells using the CD34 Microbead Kit Ultrapure and LS columns (Miltenyi Biotec) according to the manufacturer’s instructions. Colony forming unit assays were performed using methylcellulose containing media (MethoCult H4434 Enriched and MethoCult SF H4636, StemCell Technologies) according to the manufacturer’s instructions. After 14 days in culture, cells were analyzed immunophenotypically via flow cytometry.

Hislop J., Alavi A., Song Q., Schoenberger R., Kamyar K.F., LeGraw R., Velazquez J., Mokhtari T., Taheri M.N., Rytel M., de Sousa Lopes S.M., Watkins S., Stolz D., Kiani S., Sozen B., Bar-Joseph Z, & Ebrahimkhani M.R. (2023). Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering. bioRxiv.

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Hematopoietic Stem Cell CFU Assay

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The hematopoietic CFU assay was performed using serum-free MethoCult SF H4636 (STEMCELL Technologies) following the manufacturer's instructions.

Cao X., Yakala G.K., van den Hil F.E., Cochrane A., Mummery C.L, & Orlova V.V. (2019). Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives. Stem Cell Reports, 12(6), 1282-1297.

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