Recombinant human stem cell factor

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, Germany

Recombinant human stem cell factor is a protein that plays a critical role in the regulation and maintenance of hematopoietic stem cells. It is a key growth factor that stimulates the proliferation and differentiation of various hematopoietic cell lineages.

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24 protocols using recombinant human stem cell factor

Human LAD2 Mast Cell Culture

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Human LAD2 mast cell line (from D. Metcalfe, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD) was cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 ng/ml recombinant human stem cell factor (PeproTech). The cells were hemi-depleted each week with fresh medium and maintained at 0.25–5 × 10⁵ cells/ml at 37°C and 5% CO₂.

Shade K.T., Platzer B., Washburn N., Mani V., Bartsch Y.C., Conroy M., Pagan J.D., Bosques C., Mempel T.R., Fiebiger E, & Anthony R.M. (2015). A single glycan on IgE is indispensable for initiation of anaphylaxis. The Journal of Experimental Medicine, 212(4), 457-467.

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Culturing Human Mast Cells

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LAD2 (Laboratory of Allergic Diseases 2) human mast cells were cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 μg/ml streptomycin, and 100 ng/ml recombinant human stem cell factor (Peprotech). The cell suspensions were seeded at a density of 0.1 × 10⁶ cells/ml and maintained at 37°C and 5% CO₂, and periodically tested for the expression of CD117 and FcεRI by flow cytometry. Cell culture medium was hemi-depleted every week with fresh medium.

McNeil B.D., Pundir P., Meeker S., Han L., Undem B.J., Kulka M, & Dong X. (2014). Identification of a mast cell specific receptor crucial for pseudo-allergic drug reactions. Nature, 519(7542), 237-241.

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Isolation and Culture of CD34+ Cells from CML Patients

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Bone marrow samples were collected from 11 cases of newly diagnosed CML patients at the Department of Hematology of the Second Hospital of Dalian Medical University. Patients were diagnosed according to French–American–British classification. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Medical Science Ethic Committee of Dalian Medical University. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep, and cryopreserved. In addition, 5 potential donors for allogeneic bone marrow transplantation were used to purify healthy hematopoietic cells. Human CD34⁺ cells were enriched from bone marrow mononuclear cells using MiniMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [27 (link)]. Confirmation of CD34⁺ cells phenotype and purity was assessed by flow cytometry analysis using CD34-PE-Cy7 (BD Biosciences, San Diego, CA). Purified CD34⁺ cells were grown in serum-free hematopoietic growth medium (HPGM; Lonza) supplemented with 10 ng/mL recombinant human interleukin 3 (rhIL-3), 10 ng/mL rhIL-6, and 50 ng/mL recombinant human stem cell factor (PeproTech) in a humidified incubator at 37 °C and 5% CO₂/95% air (v/v).

Yan J.S., Yang M.Y., Zhang X.H., Luo C.H., Du C.K., Jiang Y., Dong X.J., Wang Z.M., Yang L.X., Li Y.D., Xia L, & Lu Y. (2022). Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells. Cell Death & Disease, 13(4), 384.

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Enrichment and Proliferation of CD34+ Cells

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Patient samples were obtained from the Manchester Cancer Research Centre Biobank (HTA 30004) and had ethical approval from the National Research Ethics Service committee (14/LO/0489). Experimental details are available in Supplementary Methods. CD34⁺ cells were enriched using CliniMACS (Miltenyi Biotec, Bisley, UK) and colony-forming assays performed in methylcellulose complete media (R&D Systems, Abingdon, UK) supplemented with 2 U/ml erythropoietin at a density of 3000 cells per ml. To assess retention of self-renewal capacity, the resulting colonies at day 7 were replated in methylcellulose and colonies counted at 14 days. CD34⁺ cells were stained using CellTrace Violet Cell Proliferation Kit (Molecular Probes, Thermo Fisher) and then seeded at a density of 1.5 × 10⁵/ml in Iscove’s modified Dulbecco’s medium, 20% (v/v) fetal calf serum, recombinant human interleukin-3 (20 ng/ml), recombinant human stem cell factor (50 ng/ml) and Flt-3 (Fms-related tyrosine kinase 3) ligand (10 ng/ml) (PeproTech, London, UK). On day 0 and following 8 days of culture, cells were harvested and stained with CD34-APC (eBioscience, Thermo Fisher) and appropriate controls using standard protocols. Fluorescence was measured on a LSRFortessa (Becton Dickenson, Oxford, UK) flow cytometer. Results were analysed using FloJo software (Ashland, OR, USA).

Pearson S., Williamson A.J., Blance R., Somervaille T.C., Taylor S., Azadbakht N., Whetton A.D, & Pierce A. (2017). Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches. Leukemia, 31(12), 2717-2725.

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HUDEP2 Cell Culture Protocol

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HUDEP2 cells were maintained with StemSpan SFEM medium (STEMCELL Technologies) supplemented with recombinant human stem cell factor (50 ng/ml; PeproTech, 300-07), recombinant human erythropoietin (20 ng/ml; PeproTech, 100-64), dexamethasone (1 μM; Sigma-Aldrich, D8893), and doxycycline hydrochloride (1 μg/ml; Sigma-Aldrich, D3072) (29 (link)).

Park S.H., Cao M., Pan Y., Davis T.H., Saxena L., Deshmukh H., Fu Y., Treangen T., Sheehan V.A, & Bao G. (2022). Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing. Science Advances, 8(42), eabo7676.

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Stem Cell Culture Optimization

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Modified Eagle’s medium (MEM), StemPro-34 SFM Medium and trypsin were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Sciencell (Carlsbad, CA, USA). Recombinant human stem cell factor was purchased from Peprotech (Rocky Hill, NJ, USA). Compound 48/80 (C48/80), l-glutamine, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-nylte-trazolium bromide (MTT), p-nitrophenyl-N-acetyl-β-d-glucosaminide and poly-d-lysine hydrobromide (PDL) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Triton X-100 was obtained from Amresco (Solon, OH, USA). Penicillin and streptomycin were purchased from Beyotime Biotechnology (Shanghai, China). Ginsenoside Rd, Rg1, Rf, 20(S)-Rg3 and 20(R)-Rg3 were purchased from Must Bio-technology (Chengdu, China). Pluronic acid F-127 was obtained from Invitrogen (Carlsbad, CA, USA). A calcium 5 assay kit was obtained from molecular devices (Sunnyvale, CA, USA). ELISA kits for histamine were purchased from Biocalvin (Suzhou, China). An Annexin V-FITC fluos staining kit was purchased from KeyGEN BioTECH (Nanjing, China).

Wang L., Zhao Y., Yang Y., Hu Y., Zou X., Yu B, & Qi J. (2017). Allergens in red ginseng extract induce the release of mediators associated with anaphylactoid reactions. Journal of Translational Medicine, 15, 148.

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Expansion and Differentiation of CD34+ Cells

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PB-CD34⁺ cells (4 × 10⁴ cells/mL) were incubated with rhTPO or 2R13 in serum-free expansion medium (SFEM, Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem cell factor (25 ng/mL; PeproTech), recombinant human IL-6 (10 ng/mL; PeproTech), recombinant human IL-9 (10 ng/mL, PeproTech), and LDL (25 μg/mL, Stem Cell Technologies for up to 14 days. Next, the cells were harvested at each time point (days 4, 7, 11, and 14) and labeled with anti-CD41a-FITC-conjugated antibody (Miltenyi Biotec), anti-CD42b-PE-conjugated antibody (Miltenyi Biotec), or 7-amino-actinomycin D (AAD; eBioscience, San Diego, CA, US) at 4 °C for 30 min. After washing with PBS, the cells were analyzed using flow cytometry.

Shin J., Kim M.J., Quan X., Kim J.W., Lee S., Park S., Jeong J.Y, & Yea K. (2023). Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia. BMC Cancer, 23, 490.

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Expansion of Primary Mast Cells

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Flow sorted primary mast cells were maintained in human mast cell culture media (StemPro^TM-34 Serum-Free media (Gibco), StemPro-34 Nutrient Supplement (Gibco), L-Glutamine (2 mM) (Gibco), Penicillin (100 U/mL)/Streptomycin (100 µg/mL) (Gibco), recombinant human stem cell factor (100 ng/mL) (PeproTech), recombinant human Interleukin 6 (100 ng/mL) (PeproTech) and Recombinant human Interleukin-3 (30 ng/mL) (PeproTech)) at 37 °C in 5% CO₂, with media changes every 5–7 days [22 (link)]. Primary mast cell numbers were determined with TC-20^TM Automated Cell Counter system (Bio-Rad) according to manufacturer’s protocol at every media change. Growth curves for primary mast cells are shown in Figure S3a.

Teng L.K., Pereira B.A., Keerthikumar S., Huang C., Niranjan B., Lee S.N., Richards M., Schittenhelm R.B., Furic L., Goode D.L., Lawrence M.G., Taylor R.A., Ellem S.J., Risbridger G.P, & Lister N.L. (2021). Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment. Cancers, 13(6), 1237.

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Induction of T, B, and Myeloid Cells from Human Intestinal Stem Cells

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Individual HSCs (Lin⁻CD45^+/dimCD34⁺CD38^−/lowCD45RA⁻CD90⁺) were sorted on a BD™ Influx cell sorter from human ileum LPLs into 96 well plates in 50 μl of complete medium, co-cultured in contact with MS5/DL1^ind100 cells in reconstituted alpha-MEM supplemented with 10% FBS (Gemini Bio-Products, Broderick, CA) and 10% human AB serum (Gemini Bio-Products, Broderick, CA) in the presence of recombinant human stem cell factor (50 ng/ml, PeproTech, Rocky Hill, NJ), rhFlt3-ligand (20 ng/ml, PeproTech, Rocky Hill, NJ), Insulin (20 nM, Sigma-Aldrich, St Louis, MO) and rhIL-7 (10 ng/ml, R&D Systems, Minneapolis, MN). Medium was half changed twice a week during the first 21 days. At 21 days, the cells were placed in fresh medium supplemented with doxycycline (1 μg/ml, Sigma-Aldrich, MO). Cultures were continued for 21 days with refreshment of medium two to three times a week. At 42 to 45 days, cells were harvested and labelled with DAPI and three anti-human antibodies (Alexa 488-conjugated mouse anti-human CD3, purified rabbit anti-human CD20 with secondary Alexa 555-conjugated donkey anti-rabbit Ab, and Alexa 647-conjugated mouse anti-human CD14) for immunofluorescent imaging analysis using a Leica DMI 6000B fluorescent microscope (Leica Microsystems, Buffalo Grove, IL).

Fu J., Zuber J., Martinez M., Shonts B., Obradovic A., Wang H., Lau S.P., Xia A., Waffarn E.E., Frangaj K., Savage T.M., Simpson M.T., Yang S., Guo X.V., Miron M., Senda T., Rogers K., Rahman A., Ho S.H., Shen Y., Griesemer A., Farber D.L., Kato T, & Sykes M. (2018). Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that are Maintained by a Circulating Pool. Cell stem cell, 24(2), 227-239.e8.

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Culturing Human Mast Cells

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LAD2 (Laboratory of Allergic Diseases 2) human mast cells were cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM l-glutamine (Fisher), 100 U/mL penicillin (Fisher), 50 µg per mL of streptomycin (Fisher) and 100 ng/mL recombinant human stem cell factor (Peprotech). The cell suspensions were seeded at a density of a million cells per mL and maintained at 37 °C and 5% CO₂.

Sbei S., Moncrief T., Limjunyawong N., Zeng Y, & Green D.P. (2023). PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain. Scientific Reports, 13, 12302.

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