Rhscf

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

The RhSCF is a laboratory instrument designed for the separation and purification of proteins. It utilizes size-exclusion chromatography to efficiently isolate target proteins from complex biological samples.

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Recombinant human stem cell factor (rhSCF; (3 citations) Recombinant human SCF (rhSCF) (2 citations)

27 protocols using rhscf

Culturing Human Mast Cell Lines

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Human mast cells (HuMCs) were obtained from peripheral blood progenitor cells using the lineage‐negative cells (CD4⁻ / CD8⁻ / CD11b⁻ / CD14⁻ / CD16⁻ / CD19⁻) which were obtained using MicroBeads (Miltenyi Biotec., Auburn, CA). The cells were cultured in METHOCULT SFBIT H4236, containing 1.2% methylcellulose with rhSCF (200 ng/mL, Peprotech, Rocky Hill, NJ), rhIL‐6 (50 ng/mL, Peprotech), and rhIL‐3 (5 ng/mL, Peprotech) 27.
The human LAD2 mastocytosis cell line was cultured in StemPro‐34 medium, with supplement, containing _L‐glutamine (2 mmol/L), penicillin (100 units/mL), streptomycin (100 μg/mL), and rhSCF (100 ng/mL, Peprotech) 28.
The HMC1.1 (expresses V560G KIT mutation) and HMC1.2 (expresses V560G and D816V KIT mutations) human mastocytosis cell lines were grown in IMDM medium supplemented with FBS (10%), _L‐glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 μg/mL) 17, 29, 30.
The K562, the 293T, the HT‐29 and the Jurkat cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA), and grown in RPMI1640 medium supplemented with FBS (10%), _L‐glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 μg/mL). The TT cell lines were also purchased from ATCC, and grown in F‐12 medium supplemented with FBS (10%), _L‐glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 μg/mL).

Ueshima C., Kataoka T.R., Takei Y., Hirata M., Sugimoto A., Hirokawa M., Okayama Y., Blumberg R.S, & Haga H. (2017). CEACAM1 long isoform has opposite effects on the growth of human mastocytosis and medullary thyroid carcinoma cells. Cancer Medicine, 6(4), 845-856.

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Expansion of Healthy and Diabetic CD34+ Cells

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CD34+ cells were prepared as previously described 10, 11, 12. The CD34+ cell fraction had a purity of 80%–90% as determined by fluorescence‐activated cell sorting (FACS) analysis. Freshly isolated CD34+ (pre‐QQc) cells from healthy and diabetic patients were placed in an ex vivo serum‐free expansion culture QQc as previously described 13. In brief, the cells were seeded at a density of 1 × 10⁴ cells per well in 24‐well Primaria plates (BD Falcon, Franklin Lakes, NJ), with 0.5 ml/well StemSpan SFEM medium (Stem Cell Tech., Vancouver, BC, Canada) supplemented with recombinant human (rh) VEGF (50 ng/ml), rhIL‐6 (20 ng/ml), rhFlt3‐ligand (100 ng/ml), rh thrombopoietin (20 ng/ml), rhSCF (100 ng/ml) (all from PeproTech, Rocky Hill, NJ), and antibiotic cocktail (Invitrogen, Carlsbad, CA), and cultured for 7 days at 37°C in 5% CO₂.

Tanaka R., Masuda H., Fujimura S., Ito‐Hirano R., Arita K., Kakinuma Y., Hagiwara H., Kado M., Hayashi A., Mita T., Ogawa T., Watada H., Mizuno H., Sawada N, & Asahara T. (2018). Quality‐Quantity Control Culture Enhances Vasculogenesis and Wound Healing Efficacy of Human Diabetic Peripheral Blood CD34+ Cells. Stem Cells Translational Medicine, 7(5), 428-438.

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Culturing Human B-ALL Cell Lines and Primary CD34+ Cells

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Human BCR-ABL1-positive B-ALL cell lines SupB15 was purchased from the American Type Culture Collection (Manassas, VA). Primary CD34⁺ cells were thawed and set up in suspension culture in IMDM medium with 20% FBS plus 100 U/ml penicillin and 100 mg/ml streptomycin in the presence of recombinant human interleukin-3 (rhIL-3, 20ng/mL, PeproTech, Rocky Hill, NJ), recombinant human interleukin-7 (rhIL-7, 20ng/mL, PeproTech, Rocky Hill, NJ), and recombinant human stem cell factor (rhSCF, 50ng/mL, PeproTech, Rocky Hill, NJ) (3-cytokine mix) [44 (link),45 (link)] at 37°C in 5% CO₂ and humidified atmosphere.

Lin X., Zou X., Wang Z., Fang Q., Chen S., Huang J., Zhe N., Yu M., Zhang Y, & Wang J. (2016). Targeting of heme oxygenase-1 attenuates the negative impact of Ikaros isoform 6 in adult BCR-ABL1-positive B-ALL. Oncotarget, 7(33), 53679-53701.

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Hematopoietic Stem Cell Expansion and Differentiation

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CD9⁺Lin⁻CD34⁺CD45RA⁻ and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs were cultured in 96-well plates with StemSpan SFEM II medium (09655, STEMCELL Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem-cell factor (rhSCF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-3 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-9 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–G colony-stimulating factor (G-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–GM colony-stimulating factor (GM-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh-thrombopoietin (TPO; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), and 20% BIT 9500 Serum Substitute (09500, STEMCELL Technologies, Vancouver, BC, Canada). Besides, 24-well Transwells (3470, Corning Incorporated, Corning, NY, USA) were used for the indirect co-culture of immune cell progenitors and CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs, and additional rhIL-7 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added in the transwell co-culture system. Lin⁻CD34⁺CD45RA⁺CD38⁺CD9⁺CD10⁺ and Lin⁻CD34⁺CD45RA⁺CD38⁺CD161⁺ were used for pre-B cells and NK/Tp enrichment, respectively (Supplemental Fig. 6d).^{68 (link)} All cultures were incubated at 37 °C in a humidified chamber under 5% carbon dioxide.

Liu Y., Zuo X., Chen P., Hu X., Sheng Z., Liu A., Liu Q., Leng S., Zhang X., Li X., Wang L., Feng Q., Li C., Hou M., Chu C., Ma S., Wang S, & Peng J. (2022). Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia. Signal Transduction and Targeted Therapy, 7, 347.

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Differentiation of Human CD34+ Cells into T Cells

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OP9 cells expressing Delta-like 4 (OP9-DL4) were maintained in α-minimum essential medium supplemented with 20% fetal bovine serum (OP9 medium) as previously described (Schmitt and Zuniga-Pflucker, 2002 (link)). A total of 3–5 × 10⁴ sorted human EB-derived CD34⁺ was added to individual wells of a 6-well plate containing OP9-DL4 cells, and cultured in OP9 medium supplemented with recombinant human (rh)FLT3L (5 ng/mL), rhIL-7 (5 ng/mL), and rhSCF (100 ng/mL) (PeproTech). Cells were passaged onto fresh OP9-DL4 cultures every 6 days.

Li Y., Brauer P.M., Singh J., Xhiku S., Yoganathan K., Zúñiga-Pflücker J.C, & Anderson M.K. (2017). Targeted Disruption of TCF12 Reveals HEB as Essential in Human Mesodermal Specification and Hematopoiesis. Stem Cell Reports, 9(3), 779-795.

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Differentiation of Primary Human Mast Cells

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Primary human mast cells were differentiated as described in^{65 (link)}. CD34⁺ hematopoietic progenitors were isolated from mobilised peripheral blood (Department of Transfusion & Tissue Medicine of the Brno University Hospital) using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec) following manufacturer recommendation.
5 × 10⁶ CD34⁺ cells were seeded at a concentration of 10⁵ cells/ml in human mast cell media (StemPRO-34 (Gibco), 100 U/ml Penicillin, 100 ug/ml Streptomycin, 1 × GlutaMAX (Gibco), 100 ng/ml rhSCF (Peprotech), 100 ng/ml rhIL6 (Stemcell Technologies)). During the first week of culture, 100 ng/ml rhIL3 (Stemcell Technologies) were added to the media. Media was changed weekly by hemi-depletion and cells were maintained at a concentration below 5 × 10⁵/ml.
Cells were used for experiments after 8 weeks of culture.

Zelante T., Paolicelli G., Fallarino F., Gargaro M., Vascelli G., De Zuani M., Fric J., Laznickova P., Kohoutkova M.H., Macchiarulo A., Dolciami D., Pieraccini G., Gaetani L., Scalisi G., Trevisan C., Frossi B., Pucillo C., De Luca A., Nunzi E., Spaccapelo R., Pariano M., Borghi M., Boscaro F., Romoli R., Mancini A., Gentili L., Renga G., Costantini C., Puccetti M., Giovagnoli S., Ricci M., Antonini M., Calabresi P., Puccetti P., Di Filippo M, & Romani L. (2024). A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis. Scientific Reports, 14, 6651.

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Liquid culture and CFC assay of CD34+ cells

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Liquid culture of CD34+ cells and CFC assay were previously described^{32 (link)}. Briefly, CD34+ were cultured in StemSpan or IMDM 5% fetal bovine serum medium (StemCell Technologies) in presence of rhSCF (100ng/ml), rhIL6 (20ng/ml), rhFlt3-L (100ng/ml) and rhTPO (20ng/ml) (all from Peprotech) for liquid culture, while for CFC assay were plated at 10³ cells/ml in semi-solid medium (MethoCult H4434 Classic - StemCell technologies) and scored by microscope 14 days later. Proliferation and mortality of liquid cultures were assessed at 3, 7, 11 and 14 days post-transduction. OP9-ΔL1 stromal cell line was kindly provided by I. Schmutz and M. Cavazzana and co-culture was performed as described for 21 days^{14 (link)}. In vitro proliferation of T cells was assessed by cell proliferation dye efluor670 (eBioscience) staining of total splenocytes or magnetically purified CD4+ and CD4+CD25- cells, where indicated, upon stimulation by antiCD3/CD28/CD2 beads (Treg suppression inspector beads – Miltenyi Biotec) following manufacturer’s instructions.

Santoni de Sio F.R., Passerini L., Valente M.M., Russo F., Naldini L., Roncarolo M.G, & Bacchetta R. (2017). Ectopic FOXP3 Expression Preserves Primitive Features Of Human Hematopoietic Stem Cells While Impairing Functional T Cell Differentiation. Scientific Reports, 7, 15820.

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Ex Vivo Expansion of CD34+ Cells

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CD34^pos cells were cultured at 10⁵ cells/mL in Stem Span^TM SFEM II supplemented with 100 ng/mL rhSCF (Peprotech, Neuilly-sur-Seine, France), 100 ng/mL rh Flt-3L (Peprotech), 20 ng/mL rhTPO (Peprotech), and 10 ng/mL rhG-CSF (Neupogen; AMGEN, Boulogne Billancourt, France). The cells were diluted in a fresh medium at days 3 and 6 to maintain the cell concentration under 1.5 × 10⁶ cells/mL, and were analyzed at the time points specified in the Results section.

Debeissat C., Avalon M., Huart M., Duchez P., Rodriguez L., Vlaski-Lafarge M., Ivanovic Z, & Brunet de la Grange P. (2022). Alpha Lipoic-Acid Potentiates Ex Vivo Expansion of Human Steady-State Peripheral Blood Hematopoietic Primitive Cells. Biomolecules, 12(3), 431.

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Differentiation of Umbilical Cord Blood Hematopoietic Stem Cells

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Umbilical cord blood (UCB) mononuclear cells were obtained and processed as previously described (La Motte-Mohs et al., 2005 (link)). They were pre-enriched into lineage-negative (Lin⁻) fractions, sorted into CD34⁺ hematopoietic stem cells, and cultured on OP9-DL1 cells for 9–10 days, as previously described (Awong et al., 2009 (link)). CD45⁺CD34⁺CD7⁺ proT-cells were FACS purified and either cultured alone (1 × 10³ cells/well) or with FOXN1-GFP⁺ (3.5 × 10³ cells/well) or GFP⁻ (3.5 × 10³ cells/well) cells in 96-well round-bottom low-attachment plates (Costar), containing APEL medium supplemented with 20% FBS, rhFLT3L (5 ng ml⁻¹, Peprotech), rhIL7 (5 ng ml⁻¹, Peprotech), and rhSCF (30 ng ml⁻¹, Peprotech). A half-media change was performed every 3–4 days. Cultures were analyzed by flow cytometry using the indicated antibodies and as detailed in the Supplemental Experimental Procedures.

Soh C.L., Giudice A., Jenny R.A., Elliott D.A., Hatzistavrou T., Micallef S.J., Kianizad K., Seach N., Zúñiga-Pflücker J.C., Chidgey A.P., Trounson A., Nilsson S.K., Haylock D.N., Boyd R.L., Elefanty A.G, & Stanley E.G. (2014). FOXN1GFP/w Reporter hESCs Enable Identification of Integrin-β4, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1+ Thymic Epithelial Progenitors. Stem Cell Reports, 2(6), 925-937.

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Murine Hematopoietic Colony Assay

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Bone marrow cells collected from 2-month-old WT mice or CD34-positive (CD34+) stem and progenitor bone marrow cells from healthy donors were seeded at a density of 5 × 10⁴ cells per 35 mm dish in semi-solid agar for GM colony growth, as previously described⁹². Cultures were incubated at 37 °C in 10% CO₂ for 10 days in presence of 10 μg/mL rhIL-3 (#200-03, Pepro Tech), 25 μg/mL rhSCF (#300-07, Pepro Tech), 25 μg/mL rhFLT3-L (#300-19, Pepro Tech), rhIL-6 (#200-06, Pepro Tech), 500 nM StemRegenin 1 (SR1; #1227633-49-9, Stem Cell Technologies) and 35 nM UM-171 (#72914, Stem Cell Technologies)^{93 (link),94 (link)}, then stained with acetyl-cholinesterase and counted for the number of colonies.

Tremblay C.S., Chiu S.K., Saw J., McCalmont H., Litalien V., Boyle J., Sonderegger S.E., Chau N., Evans K., Cerruti L., Salmon J.M., McCluskey A., Lock R.B., Robinson P.J., Jane S.M, & Curtis D.J. (2020). Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells. Nature Communications, 11, 6211.

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