Prior to differentiation, single dissociated undifferentiated human γδT‐iPSCs were seeded onto a Laminin511‐E8‐coated 6‐well plate at a density of 2 × 103 cells and cultured in Stem Fit medium. When individual colonies grew to around 500 µm in diameter, the medium was replaced by Stem Fit medium supplemented with 4 µM of CHIR99021 (Tocris Bioscience, Bristol, UK), 80 ng/ml of rhBMP4 (R&D Systems) and 80 ng/ml of rhVEGF (R&D Systems). This day was defined as day 0 of our protocol. On day 2, the medium was refreshed. On day 4, the medium was replaced by Essential 6 medium (ThermoFisher Scientific) supplemented with 4 µM of SB43152 (WAKO), 80 ng/ml of rhVEGF, 50 ng/ml of rh‐bFGF (Reprocell) and 50 ng/ml of rhSCF (R&D Systems). On day 6, the medium was replaced by StemPro‐34SFM (Thermo Fisher Scientific) supplemented with 20 ng/ml of rhVEGF, 50 ng/ml of rhSCF, 50 ng/ml of rhIL‐3 (R&D Systems), 50 ng/ml of hIL‐6 (Roche, Basel, Switzerland), 50 ng/ml of rhFlt3 ligand (R&D Systems) and 10 IU/ml of recombinant human Erythropoietin α (EPO) (Kyowa Hakko Kirin, Tokyo, Japan). On day 8, the medium was replaced by StemPro‐34SFM supplemented with 50 ng/ml of rhSCF, 50 ng/ml of hIL‐6 (Roche) and 10 IU/ml of EPO. On day 10, the medium was refreshed. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Rhscf
Manufactured by R&D Systems
Sourced in United States
RhSCF is a recombinant human stem cell factor. It is a soluble protein that supports the growth and differentiation of various cell types, including hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Imatinib, by Abcam (3 mentions)
Interleukin 3 (il 3), by R&D Systems (3 mentions)
Rhil 3, by R&D Systems (2 mentions)
Rhvegf, by R&D Systems (2 mentions)
Rhbmp 4, by R&D Systems (2 mentions)
Retronectin, by Takara Bio (2 mentions)
Flt3l, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Sp600125, by Merck Group (2 mentions)
U0126, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (1 mentions)
Essential 6 medium, by Thermo Fisher Scientific (1 mentions)
Hil 6, by Roche (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
U0126, by Abcam (1 mentions)
Rat anti c kit, by Thermo Fisher Scientific (1 mentions)
Thrombopoietin, by R&D Systems (1 mentions)
11 protocols using rhscf
1
Directed Differentiation of γδT-iPSCs into Hematopoietic Cells
2
Modulating SCF/KIT Signaling in HT-29 Cells
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To activate the SCF/KIT signalling, we constructed lentivirus vector containing CDS of KIT gene by the technical support from GeneChem (Shanghai, China) and transfected it into HT-29 cells. After stably over-expressing KIT, exogenous rhSCF (50 ng/ml, R&D) was added into medium for 36 h. To inactivate the SCF/KIT signalling, 2 μM Imatinib (BioVision) was used for 36 h.
3
Evaluating c-kit Signaling Pathway
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The following reagents were used: rabbit anti-c-kit (CST, USA), rabbit anti-phospho-c-kit (CST, USA), rabbit anti-Erk1/2 (CST, USA), rabbit anti-phospho-Erk1/2 (CST, USA), rat anti-c-kit (eBioscience, USA), mouse anti-ETV5 (Santa Cruz, USA), G-1™ (Vitrolife, Sweden), G-2™ (Vitrolife, Sweden), Human serum album (HSA) solution (Vitrolife, Sweden), Imatinib (Biovision, USA), and recombinant human SCF (rhSCF, R&D Systems, USA), U0126 (Biovision, USA).
4
Generation of Leukemic Humanized Mice
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Pseudo-typed retroviruses were produced by transfection using Lipofectamine 2000 (Invitrogen, San Diego, CA) of 293FT cells with a 3-plasmid system consisting of the MLL fusion vector (MSCV-MLL-AF9-human pgk-EGFP; kindly provided by Dr. John Dick) (Barabe et al., 2007 (link)) and two packaging vectors (VSV-G and Psi-Env). Viral particles were collected 48 or 72 h post-transfection and concentrated by ultracentrifugation at 50,000 g for 2 h, and were titered and stored at − 80 °C until use. For transduction, human CD34+ FLCs were stimulated overnight in medium containing 50 ng/mL rhSCF (R&D, Minneapolis, MN), 50 ng/mL Flt-3L (eBioscience, San Diego, CA), 25 ng/mL TPO (R&D, Minneapolis, MN), 10 ng/mL IL-3 (R&D, Minneapolis, MN), in 24-well plates pre-coated with Retronectin (Takara Bio Inc.), followed by incubation with retroviruses for 12 h. Cells were washed twice and injected into sublethally irradiated NSG mice to generate leukemic hu-mice (see Hu-mouse preparation below). A small aliquot of the transduced cells was cultured for 3 additional days to determine the transduction efficiency by measuring the ratio of GFP+ cells using FACS (ranged between 10 and 30% in the experiments presented).
5
Granulocyte and Mast Cell Isolation
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Granulocytes from PB and BMMCs were purified using Biocoll Separation Solution 1.077 g/ml (Biochrom, Berlin, Germany). Eosinophils were purified by negative selection using CD16 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Granulocytes were cultured at a density of 5 × 105 cells/ml media containing RPMI 1640 (Life Technologies, Carlsbad, USA), 20% FBS Good Forte (PAN Biotech, Aidenbach, Germany), 25 mM HEPES, 2 mM L-glutamine, 1 × MEM NEAA, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol (all from Life Technologies, Carlsbad, USA) and 10 ng/ml rhIL-5 (Biolegend, San Diego, USA). BMMCs were kept at a density of 5 × 105 cells/ml in media containing 20% BIT 9500 (StemCell, Vancouver, Canada), 100 ng/ml rhSCF, 5 ng/ml interleukin-6, 10 ng/ml interleukin-3, 10 ng/ml thrombopoietin (all from R&D Systems, Minneapolis, USA), 10 µM β-mercaptoethanol and 4 µg/ml low-density lipoproteins (StemCell, Vancouver, Canada).
6
Lentiviral Transduction of Human CD34+ Cells
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Pseudotyped lentiviral vectors were produced by transfection using Lipofectamine 2000 (Invitrogen, San Diego, CA) of 293FT cells in 10-cm plates. a 4-plasmid system consisting of the transfer vector (MART-1 antigen specific TCR, DMF5 clone; 10μg) and 3 packaging plasmids (VSV-G 3.6μg, pMDLg/pRRE 6.7 μg, and pRSV-Rev 6.7 μg) was used for MART-1 TCR-lentivirus production. The supernatant was collected 48 hour post-transfection and concentrated by ultracentrifugation at 50,000 g for 2 hours. Lentiviruses were stored at −80°C until use. Human CD34+ FLCs were stimulated overnight in media containing 50ng/mL rhSCF (R&D, Minneapolis, MN), 50ng/mL Flt3L (ebioscience, San Diego, CA), 25 ng/mL TPO (R&D), and 10ng/mL IL-3 (R&D), in a 24-well plate pre-coated with retronectin (Takara Bio Inc), followed by transduction with lentiviral vectors for 12 hours. Cells were washed twice and intravenously injected into sub-lethal irradiated mice as described above.
7
Investigating SCF/c-KIT Signaling Pathways
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The human CRC-derived LS174T cell line was purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, YN, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco) at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Cells were treated with recombinant human SCF (rhSCF) (50 ng/mL; R&D System, Minneapolis, MN, USA) for 12, 24, or 36 h in order to activate c-KIT, While Imatinib (2 μM; BioVision, Zurich, Switzerland) was used to block SCF/c-KIT signaling activity. After serum starvation overnight, AKT, JNK, and ERK signaling was inhibited by MK2206 2HCL (5 μM; Selleck Chemicals, Shanghai, China), SP600125 (10 μM; Sigma-Aldrich), U0126 (10 μM; Sigma-Aldrich respectively), and rhSCF (50 ng/mL) was added 1 h after inhibitor treatment. Cells were harvested 24 h later.
8
Investigating SCF and TPA Signaling in CRC Cells
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The human CRC cell lines, HT-29 and DLD-1 cells, and normal HEK293 cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in Dulbecco's modified eagle’s medium (DMEM ) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) at 37 °C in the presence of 5% CO2.
The cells were treated with recombinant human SCF (rhSCF) (R&D System, Minneapolis, MN, USA) in a gradient of concentrations (0, 25, 50, 100 ng/mL) or for different time (0, 6, 12, 24, 36, 48 h) or phorbol 12-O-tetradecanoate-13-acetate (TPA, 20 nM; Sigma-Aldrich) for different times (3, 6, 12, 24 h). After serum starvation overnight, rhSCF (50 ng/mL) was added into the medium 1 h after specific inhibitor treatment including SP600125 (JNK inhibitor, 10 µM; Sigma-Aldrich), SB203580 (p38 inhibitor, 10 µM; Sigma-Aldrich) and U0126 (extracellular signal regulated kinase (ERK) inhibitor, 10 µM; Sigma-Aldrich), respectively. The cells were harvested 36 h later and used for further experiments.
The cells were treated with recombinant human SCF (rhSCF) (R&D System, Minneapolis, MN, USA) in a gradient of concentrations (0, 25, 50, 100 ng/mL) or for different time (0, 6, 12, 24, 36, 48 h) or phorbol 12-O-tetradecanoate-13-acetate (TPA, 20 nM; Sigma-Aldrich) for different times (3, 6, 12, 24 h). After serum starvation overnight, rhSCF (50 ng/mL) was added into the medium 1 h after specific inhibitor treatment including SP600125 (JNK inhibitor, 10 µM; Sigma-Aldrich), SB203580 (p38 inhibitor, 10 µM; Sigma-Aldrich) and U0126 (extracellular signal regulated kinase (ERK) inhibitor, 10 µM; Sigma-Aldrich), respectively. The cells were harvested 36 h later and used for further experiments.
9
Expansion of Hematopoietic Stem Cells
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Sorted primary murine cKit+ and LSK cells were cultured in SFEMII media (Stemcell technology) supplemented with; rhFlt3/Flk-2 ligand (Stemcell technology), rhTPO (Stemcell technology), rhIL-6 (R&D system), rmIL-3 (R&D systems) and rmSCF (R&D systems) at a concentration of 20 ng/mL. UCB samples were provided by the Karolinska Hospital (Stockholm, Sweden) with informed consent from the parents and all investigation has been performed according ethical standards and to the declaration of Helsinki and to national and international guidelines. The experiments on the UCB samples were approved by The Regional Ethical Review Board in Stockholm (Dnr: 2012/480-31/1).
Lymphoprep solution (Invitrogen) was used to isolate mononuclear cell fraction and CD34+ cells were enriched by using CD34 microbead kit (Miltenyi Biotec). The CD34+ and sorted HSC UBC cells were expanded in SFEMII media supplemented with; rhIL-6, rhIL-3 (R&D systems), rhFl3/Flk-2 ligand, rhTPO and rhSCF (R&D systems) all in final concentrations of 20 ng/mL.
Lymphoprep solution (Invitrogen) was used to isolate mononuclear cell fraction and CD34+ cells were enriched by using CD34 microbead kit (Miltenyi Biotec). The CD34+ and sorted HSC UBC cells were expanded in SFEMII media supplemented with; rhIL-6, rhIL-3 (R&D systems), rhFl3/Flk-2 ligand, rhTPO and rhSCF (R&D systems) all in final concentrations of 20 ng/mL.
10
Directed Hematopoietic Differentiation of Human Pluripotent Stem Cells
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hPSCs (NIH Codes: WA01, H1) were maintained on irradiated CF1 feeder cells as described in our previous paper (20 (link)). Before lentiviral transduction and hematopoietic differentiation, hPSCs were maintained in chemically defined mTeSR™1 medium (StemCell Technologies, Canada) without feeder as described previously (21 (link)). The 293T cells were maintained in DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA). GFP-labeled OP9 (OP9-GFP) cells were maintained in α-MEM medium (Gibco, USA) supplemented with 20% FBS (Gibco) (12 (link)). hPSCs (H1 or H1+TFs) monolayers at 70% confluence were cultured in X-Vivo 15 medium (Lonza, Switzerland) supplemented with 1 μg/mL doxycycline (Dox), 1 mM sodium pyruvate (Sigma-Aldrich, USA), 1x non-essential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 mM 2-mercaptoethanol (Sigma-Aldrich), and the four growth factors, including 50 ng/mL recombinant human bone morphogenetic protein-4 (rhBMP-4, R&D, USA), 50 ng/mL recombinant human vascular endothelial growth factor (rhVEGF, R&D), 50 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF, R&D), and 20 ng/mL recombinant human stem cell factor (rhSCF, R&D). After a 4-day culture, cells were collected for flow cytometry analysis.
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