GPC were obtained by the protocol reported for Rezania et al. [15 (link)] with modifications. The protocol consists of 6 stages: stage 1, incubated for 4 days with RPMI 1640 (Gibco™. Catalog number 31800), bovine serum albumin (BSA) (Sigma-Aldrich, catalog number A9418) 2%, activin A 100 ng/mL (Prospecbio), Wnt3a 20 ng/mL (Sigma-Aldrich. Catalog number H17001), FGF2 8 ng/mL (Prospecbio. Catalog number CYT-085). Stage 2, incubated for 2 days with DMEM-F12 medium with BSA 2%, FGF7 50 ng/mL (Prospecbio. Catalog number CYT-303), Cyclopamine-KAAD 0.25 µM (SCBT, Dallas, Tx, USA. Catalog number sc-200929). Stage 3, incubation for 4 days with DMEM-F12 medium, Cyclopamin-KAAD 0.25 µM, retinoic acid (RA) 2 µM (Sigma-Aldrich. Catalog number R2625), FGF7 50 ng/mL (Prospecbio), Noggin 100 ng/mL (Prospecbio. Catalog number CYT-475). Stage 4, incubation for 3 days with DMEM:F12 medium, Inhibitor II ALK5 1 µM (SCBT. Catalog number sc-221234), Noggin 100 ng/mL, DAPT 1 µM (SCBT. Catalog number sc-201315). Stage 5, incubation for 7 days with DMEM/F12 medium, inhibitor II ALK5 1 µM. Stage 6, incubation for 7 days with DMEM F12 medium. However, we used 2% v/v FBS instead of 1% B27 as a supplement to differentiation medium during stages 3 to 6, Fig. 1 b.
Noggin
Manufactured by ProSpec
Sourced in United States
Noggin is a versatile piece of lab equipment designed for use in various scientific and research applications. It serves as a compact and portable platform for experimental setup and data collection. The core function of Noggin is to provide a stable and adjustable base for mounting and positioning laboratory instruments, samples, or other components as required by the research or experimental procedure.
Automatically generated - may contain errors
Lab products found in correlation
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Retinoic acid, by Merck Group (1 mentions)
Wnt3a, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by ProSpec (1 mentions)
Activin a, by ProSpec (1 mentions)
Hydrocortisone, by Bio-Techne (1 mentions)
Methyl 3 2 4 2 adamantyl phenoxy acetyl amino 4 hydroxybenzoate, by Santa Cruz Biotechnology (1 mentions)
Trilencer 27 mouse sirna, by OriGene (1 mentions)
Recombinant murine hgf, by Thermo Fisher Scientific (1 mentions)
Human bfgf, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
A83 01, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Welgene (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
3 protocols using noggin
1
Differentiation of Pancreatic Progenitors
2
Integrin and Cell Signaling Modulation
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Integrin-blocking antibodies α5β1 (Millipore) and αvβ3 (Santa Cruz Biotechnology) were added to cells at 1 μg/ml in medium before seeding and after changing the medium at 1 μg/ml. MAPK inhibitors [FR180204 (ERK1/2), SP600125 (JNK), and SB202190 (p38); 6 μM; Calbiochem], cytoskeletal inhibitor [blebbistatin (1 μM) and Y27632 (2 μM); Calbiochem], BMP inhibitor (Noggin; 5 ng/ml; ProSpec), GSK-3 inhibitor (CHIR; 10 nM; Calbiochem), HIF1 inhibitor (methyl 3-[[2-[4-(2-adamantyl)phenoxy]acetyl]amino]-4-hydroxybenzoate; 10 nM; Santa Cruz Biotechnology), hydrocortisone (0.5 mM; Tocris), and HSS (0.1 mg/ml; Tocris) were added to the medium after cell seeding with the first medium change. For inhibition assays, B16F0s were cultured for 3 days with the inhibitors, washed twice with PBS, and then cultured with fresh medium without the inhibitors to prevent the addition of the drugs in the conditioned medium to hMVECs.
siRNAs for Jarid1B (ID: 75605; Trilencer-27 Mouse siRNA) or scrambled siRNAs were purchased from OriGene. Transfection was performed according to the vendor’s instructions. Lipofectamine 2000 was used for transfection (100 nM). As with inhibition assays, cells were washed twice with PBS at day 3 and cultured with fresh medium to be conditioned.
siRNAs for Jarid1B (ID: 75605; Trilencer-27 Mouse siRNA) or scrambled siRNAs were purchased from OriGene. Transfection was performed according to the vendor’s instructions. Lipofectamine 2000 was used for transfection (100 nM). As with inhibition assays, cells were washed twice with PBS at day 3 and cultured with fresh medium to be conditioned.
3
Tonsil Organoid Culture Protocol
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The tonsil samples were chopped and washed with Dulbecco's phosphate-buffered saline (D-PBS; Welgene, Daegu, Korea) and then enzymatically digested with 1 mg/mL collagenase II (Gibco) in advanced DMEM/F12 (Gibco) for 2 h at 37 °C. After digestion, isolated cells were embedded in Matrigel (Corning Inc., Corning, NY, USA) in a 48-well plate (SPL Inc., Seongnam, Korea) and incubated at 37 °C for 10 min to polymerize the matrices. Tonsil organoids were cultured in advanced DMEM/F12 supplemented with Antibiotic–Antimycotic, Glutamax (Thermo Fisher Scientific), B27 (Invitrogen, Carlsbad, CA, USA), 10% conditioned media from Cultrex HA-R-spondin1-Fc 293T cells (R&D Systems, Minneapolis, MN, USA), and the following growth factors: 50 ng/mL recombinant murine HGF (Peprotech, Rocky Hill, NJ, USA), 100 ng/mL noggin (ProSpec, St. Paul, MN, USA), 20 nM A83-01 (Sigma), 50 ng/mL human FGF10 (ATGen, Seongnam, Korea), 20 ng/mL human bFGF (Peprotech), 10 μM prostaglandin E2 (BioGems, Westlake Village, CA, USA), and 10 mM nicotinamide (Sigma). For passage, organoids were dissociated by incubation in 0.25% trypsin-EDTA (Invitrogen, Waltham, Massachusetts, USA) every 7–10 days depending on the number and size of organoids. For the first 2 days at every passage, 10 μM Y-27632 (Tocris Biosciences, Bristol, UK) was added to the culture medium.
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