For PGCLC induction, cells were dissociated by incubation with Accutase and then transferred into a low-cell-binding U-bottom 96-well plate (Greiner, #650970) in GK15 medium (GMEM (Thermo, #11017035) containing 15% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate (Thermo, #11360070), 2 mM GlutaMax, 0.1 mM 2-mercaptoethanol, and 1 × penicillin/streptomycin) supplemented with 200 ng/ml human BMP4 (R&D Systems, #314-BP-01 M), 100 ng/ml mouse SCF (R&D Systems, #455-MC-500), 1000 U/ml human LIF (Wako, #125-06661), 50 ng/ml human EGF (R&D Systems, #236-EG) and 10 μM ROCK inhibitor with or without 2.5 μM IWR1 (TOCRIS, #3532) and 1.0 µM BMS493 (R&D Systems, #3509/10). In each well, 5 × 103 cells were used. In the experiments requiring enforced expression of the transgenes, 100 μM dexamethasone, 0.5 μg/ml doxycycline, and 0.5 μM Shield-1, which induce SOX17, BLIMP1, and TFAP2C expression, respectively, were added to the medium11 (link). To enforce the expression of SOX17 alone, only 100 μM dexamethasone was added.
Bms493
Manufactured by R&D Systems
BMS493 is a synthetic compound that functions as a retinoic acid receptor (RAR) antagonist. It inhibits the transcriptional activity of RARs, which are nuclear receptors that play a role in regulating gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Human egf, by R&D Systems (1 mentions)
Human bmp4, by R&D Systems (1 mentions)
Mouse scf, by R&D Systems (1 mentions)
Human lif, by Fujifilm (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Etomoxir, by Bio-Techne (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Recombinant mouse il 33, by BioLegend (1 mentions)
3 protocols using bms493
1
Induction of PGCLC from Dissociated Cells
2
Modulating Metabolic Pathways in Mice
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To block RA signaling, a total of 220 mg of the pan RA receptor inverse agonist BMS 493 (R&D Systems; RAi) was resuspended in 30 µl of biotechnology performance–certified DMSO (Sigma-Aldrich) and administered intraperitoneally to C57BL/6 WT mice every day for 8 d as previously described (Kastner et al., 2001 (link)). To block FAO, mice were administered 15 mg/kg etomoxir (Tocris Bioscience) every other day throughout the course of infection or for 7 d under steady-state conditions. To block glycolysis, mice were administered 1 g/kg 2-DG (Cayman Chemicals) every other day starting upon infection with T. muris for 7 d and then every day thereafter. For simultaneous treatment, mice were treated with the indicated combination in a total volume of 30 µl DMSO or DMSO only as a vehicle control. To block FA uptake, mice were treated with 200 mg/kg orlistat every other day starting 7 d after infection. Control mice received DMSO only.
3
IL-33 Induced ILC2 Activation and Expansion
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For ILC2 activation and expansion, mice received injections of 200 or 400 ng recombinant mouse (rm) IL-33 (BioLegend) in 200 μl PBS i.p. on four consecutive days. Controls received 200 μl PBS. ILC2s were isolated at 2–3 wk after the first IL-33 injection. To block RA signaling, a pan-RAi (BMS493; R&D) was dissolved in DMSO (final conc. 29.3 µg/μl). 220 µg RAi/animal (diluted in PBS) was administered i.p. every second day. The same DMSO concentration was administered to control animals.
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