Human endometrial stromal cells (hESCs) from normal endometrial biopsies were dissected and digested according to the method described above and then filtered through a 40‐μm cell strainer to separate the stromal cells from the glands. Cells were inoculated in DMEM/F12 (Wisent Inc) containing 10% FBS and cultured at 37°C with 5% CO2 and saturated humidity. The hESCs from passages 2–4 were used for all experiments. Chemicals and recombinant proteins used in the article were recombinant human Gas6 (order no. 885‐GSB, R&D Systems), Bemcentinib (order no. HY‐15150, MedChemExpress), and JSH23 (order no. HY‐13982, MedChemExpress).
Bemcentinib
Manufactured by MedChemExpress
Sourced in United States
Bemcentinib is a laboratory chemical compound used for research purposes. It is a small molecule inhibitor. The core function of Bemcentinib is to inhibit a specific biological target, though its intended use is not provided in this factual description.
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Lab products found in correlation
Jsh 23, by MedChemExpress (1 mentions)
Dmem f12, by Wisent (1 mentions)
Y 27632, by Cell Signaling Technology (1 mentions)
Bms 536924, by MedChemExpress (1 mentions)
Erdafitinib, by MedChemExpress (1 mentions)
Anti gas6 antibody, by R&D Systems (1 mentions)
Ly294002, by Merck Group (1 mentions)
Lapatinib, by MedChemExpress (1 mentions)
Anti axl antibody, by R&D Systems (1 mentions)
Gefitinib, by MedChemExpress (1 mentions)
Bobcat339, by MedKoo Biosciences (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Corning (1 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Low density lipoprotein ldl, by STEMCELL (1 mentions)
4 protocols using bemcentinib
1
Isolation and Culture of Human Endometrial Stromal Cells
2
Kidney Allograft Tolerance in Mice
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Kidneys from 6- to 7-week-old BALB/c (male or female) mice were transplanted into bilaterally nephrectomized B6 (WT or KO) male recipients. In some of the experiments, kidney allograft tolerance was induced in the recipients by infusions of donor apoptotic cells. In brief, BALB/c SP were treated with ECDI (Calbiochem, 150 mg/mL per 3.2 × 108) on ice for 1 hour with agitation and washed. A total of 1 × 108 BALB/c ECDI-SP cells was infused to the B6 recipient i.v. on d–7 and d+1, while d0 was designated as the day of transplantation (27 (link)). In additional experiments, WT B6 recipients were further treated with bemcentinib, a selective inhibitor of Axl kinase activity (MedChemExpress). Recipients received daily oral gavage of bemcentinib (100 mg/kg/day) from d–1 to d+3. To investigate donor antigen–specific T cell responses, 2.5 × 105 purified TCR75 CD4+ T cells were adoptively transferred into WT or KO B6 recipients 1 day prior to the first infusion of donor apoptotic cells. In some of the experiments, congenic CD45.1 B6 mice were used as recipients for kidneys from CD45.2 BALB/c donors.
3
RTK inhibition and AXL signaling in U2OS cells
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Receptor tyrosine kinases (RTKs) were inhibited in U2OS cells overnight with 1 μM of each of the following inhibitors: Gefitinib (#HY-50895), Lapatinib (#HY-50898), Bemcentinib (#HY-15150), BMS-536924 (#HY-10262) purchased from MedChemExpress; Erdafitinib (#HY-18708) Lenvatinib (#HY-10981), Cabozantinib (#HY-13016), Galunisertib (#HY-13226), Linsitinib (#HY-10191) from Cedarlane Laboratories; and Crizotinib (#PF-02341066) from Selleckchem.com. PI3K was inhibited by treating cells with LY294002 (Millipore Sigma; #440202) at 10 μM for at least 1 h. Gas6-mediated stimulation of AXL in U2OS cells was performed by serum-starving cells for 24 hours prior to the addition of 200 ng/mL recombinant Human Gas6 (R&D Systems; #885-GSB) to the culture medium for 30 min. For AXL receptor blockade, U2OS cells were incubated with 20 μg/mL anti-AXL antibody (R&D Systems; #AF154) or 50 μg/mL anti-GAS6 antibody (R&D Systems; #AB885). Normal goat polyclonal IgG (R&D Systems; #AB-108-C) was used as control (50 μg/mL). Rho-associated kinases (ROCK) were inhibited with Y-27632 (Cell Signaling Technology; #13624) at 1 or 5 μM.
4
Expansion of Hematopoietic Stem Cells
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Pooled CD34+ cells (two donors) were cultured in 24-well plate (Corning, New York, United States) in serum-free medium (SFM) StemSpan SFEM (StemCell Technologies) supplemented with stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3L) each at 100 ng/ml, low-density lipoprotein (LDL, Stem cell Technologies) at 10 μg/ml and 1% Penicillin-Streptomycin (Gibco) at a concentration of 12000 cells/mL. All cytokines were purchased from Peprotech (Rocky Hill, NJ, USA). This medium is referred to as STFL or control. Cultures were maintained at 37°C, 5% CO2 with fresh medium added on day 4, 7 and 10. Cell expansion was monitored on day 7 and 14 by cytometric analysis. Absolute number of stem and progenitor cells were determined on day 7 and 14, and net fold expansions were derived by dividing net final cell numbers by the starting cell numbers. In indicated cultures, 30uM BobCat-339 (Cat# 408006, Medkoo Biosciences, NC, USA), 2uM Bemcentinib (MedChemExpress LLC, Cat# HY-15150 NJ, USA), 10 ng/mL neutralizing GAS6 antibody (Cat# AB885, R&D systems, MIN, USA), recombinant GAS6 (R&D Systems, cat# 885-GSB-050, optimal concentration determined at 10 ng/mL) or lentivirus (MOI 10) were added on day 10 and cultured for 4 days. On day 14, all the cells were harvested and analyzed for cell expansion, phenotype, viability and apoptosis and cell cycle analysis.
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