Activin A and TGFβ1 were reconstituted in PBS plus 4 mM HCl according to the manufacturer’s instruction (both R&D, Minneapolis, MN). Follistatin 288 (R&D) was reconstituted in PBS.
Follistatin 288
Manufactured by R&D Systems
Sourced in United States
Follistatin-288 is a glycoprotein that binds to and neutralizes the activity of activin, a member of the transforming growth factor-beta (TGF-β) superfamily. It is a natural antagonist of activin and regulates its biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Bone morphogenetic protein 4 (bmp4), by R&D Systems (3 mentions)
Oncostatin, by R&D Systems (2 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (2 mentions)
Activin a, by R&D Systems (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Matrigel, by Corning (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
Stemdiff apel medium, by STEMCELL (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Nodal, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dispase, by BD (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using follistatin 288
1
Reconstitution of Activin A, TGFβ1, and Follistatin
2
Hepatocyte Toxicity Screening Assay
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HLCs were harvested after 20 days of hepatic differentiation cells with 2× TrypLE (Life Technologies), and plated onto Matrigel (Corning)-coated 96 well plate at the density of 3 × 104 cells per well for overnight incubation. Cells were then treated with serially diluted concentrations of acetaminophen (APAP, Sigma) and pazopanib hydrochloride (Selleck Chemicals) in the basal differentiation medium supplemented with 20 ng/ml HGF (R&D systems), 100 ng/ml Follistatin-288 (R&D systems) and 20 ng/ml Oncostatin (R&D systems) for 24 hours. Drug dilutions were prepared in DMSO. Cell viability was determined by the MTS assay using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega) and the ATP assay was performed with CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega).
For viability experiments with N-acetyl cysteine (NAC), a similar procedure was adopted except cells were co-incubated with 0.5 mM NAC (Hidonac®, Zambon S.p.A.) and PZ for 24 hours. Vehicle controls were appropriately modified to include 0.05% EDTA in PBS (in which NAC was dissolved). Cell viability was measured using CellTiter-Glo reagent.
For viability experiments with N-acetyl cysteine (NAC), a similar procedure was adopted except cells were co-incubated with 0.5 mM NAC (Hidonac®, Zambon S.p.A.) and PZ for 24 hours. Vehicle controls were appropriately modified to include 0.05% EDTA in PBS (in which NAC was dissolved). Cell viability was measured using CellTiter-Glo reagent.
3
Efficient Differentiation of iPSCs into Hepatocytes
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The differentiation of iPSCs into hepatocytes was carried out as described previously35 (link) with slight modification. Briefly, iPSCs were cultured to 70% confluence before switching to a basal differentiation medium containing 100 ng/ml activin A (R&D systems) and 50 ng/ml WNT3a (R&D systems) for 6 days (step 1), followed by 10 ng/ml FGF2 (R&D systems) and 50 ng/ml BMP4 (R&D systems) in differentiation medium (half basal differentiation medium with half STEMdiff™ APEL™ medium (Stemcell Technologies)) for 4 days (step 2). On the following 4 days, 50 ng/ml FGF1 (R&D systems), 10 ng/ml FGF4 (R&D systems) and 25 ng/ml FGF8 (R&D systems) was applied in the differentiation medium (step 3). Finally, the cells were incubated with 20 ng/ml HGF (R&D systems) and 100 ng/ml Follistatin-288 (R&D systems) for 4 days with additional 20 ng/ml Oncostatin (R&D systems) for another 2 days (step 4). The medium was changed every other day during the 20-days differentiation period.
4
Esophageal Cell Lines and Treatments
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The Barrett's esophagus cell line CPB (CRL-4028) was purchased from American Type Culture Collection (ATCC) and cultured with epithelial cell medium 2 (ScienCell, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS, Hyclone, GE Healthcare, Pittsburgh, PA) and antibiotics, 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, Carlsbad, CA). The esophageal adenocarcinoma cell lines, OE33 and FLO-1, were derived by Dr. David Beer [60 (link)] and grown in RPMI and DMEM (Invitrogen, Carlsbad, CA), respectively, with 10% FBS at 37°C in 5% CO2. Fibroblasts were grown in DMEM with 5% FBS (Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco). For treatment with growth factors 5 ng/ml recombinant human TGFβ1, 10 ng/ml Activin A, 100 ng/ml Follistatin-288, 100 ng/ml Nodal (all R&D Systems, Minneapolis, MN Systems) or 1 μM A83-01 (Tocris, Bristol, UK) were used. Overexpression of Activin A (INHBA) was achieved by retroviral transfection of cells with viral supernatant containing pBABE plasmid with zeocin resistance (Addgene, Cambridge, MA) encoding the INHBA gene sequence (Origene, Rockville, MD).
5
Organotypic Skin Reconstructs: Epithelial Differentiation
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Organotypic reconstructs were grown as previously described [18 (link), 20 (link)] with the exception that each culture was rinsed in 1XPBS and incubated with Epidermalization 3 medium lacking serum for two additional days prior to harvesting. The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: 5 ng/ml recombinant human TGFβ1, 10 ng/ml Activin A, 100 ng/ml Follistatin-288 (all from R&D Systems) or 1 μM A83-01 (Tocris).
6
Organoid Culture of Murine Airway Basal Cells
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Ngfr+ BCs isolated as described (Rock et al., 2009 (link)) from C57BL/6 or Foxj1-GFP mice were suspended in MTEC/plus medium (Rock et al., 2009 (link)), mixed 1:1 with growth factor-reduced Matrigel (Corning Life Sciences), and seeded at 1000 cells/well in 24-well 0.4 μm pore inserts (#3470, Corning Life Sciences). Factors were added to the medium in the lower well, and the medium changed every other day. MTEC/SF medium (Rock et al., 2009 (link)) was used from day 7. Images were taken using an AxioVert 200M (Carl Zeiss). Spheres were counted at day 9 and dissociated using dispase (BD Biosciences, 354235; 70 µl/well at 37°C for 30 min) and 0.1% trypsin/EDTA (GIBCO, 15400-054) and cell number counted using a hemocytometer. For quantifying GFP+ cells, dissociated cells were fixed with 2% paraformaldehyde (PFA) in PBS, and analyzed using FACSCanto (BD Biosciences). For quantifying proliferation, spheres were incubated in 10 μM EdU for 2 h and staining carried out using Click-iT EdU Imaging Kit (Invitrogen). Bmp4, Bmp5, recombinant mouse Chrd and follistatin 288 were from R&D Systems. LDN-193189 was from Stemgent, recombinant mouse Nog was from PeproTech and DMH1 was from Sigma (D8946).
7
Activin, TGF-β, and BMP Signaling Evaluation
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Recombinant human Activin A, Activin B, TGF-β3, BMP6, BMP9, Follistatin 288, and ALK1-Fc were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human FSTL3 was purchased from BioLegend (San Diego, CA, USA). GDF8 and GDF11 were purchased from Pepro Tech (Rocky Hill, NJ, USA). Murine ActRIIB-murine Fc was purchased from R&D Systems, and murine ActRIIB-murine Fc/RAP-031 was kindly provided by Acceleron Pharma (Cambridge, MA, USA).
The following antibodies were used: anti-human IgG Fc (Bethyl Laboratories, Montgomery, TX, USA), anti-human FSTL3 (ab170680; Abcam, Cambridge, UK), anti-Laminin (ab11575; Abcam), and anti-BMP9 (MAB3209; R&D).
The following antibodies were used: anti-human IgG Fc (Bethyl Laboratories, Montgomery, TX, USA), anti-human FSTL3 (ab170680; Abcam, Cambridge, UK), anti-Laminin (ab11575; Abcam), and anti-BMP9 (MAB3209; R&D).
8
Ovine LH and BMP Modulation of Androstenedione
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Ovine LH (NIADDK oLH-S-16) was obtained from NHPP, Torrance, CA, USA. Recombinant human BMP2, BMP4, BMP6, BMP7, gremlin, noggin, follistatin-288 and recombinant mouse chordin were purchased from R&D Systems. Treatments were prepared in Hank's balanced salt solution containing 0.1% (w/v) BSA and sterile stock solutions prepared using 0.2 µm membrane filters before further dilution in sterile culture medium. The concentrations of LH (150 pg/mL) and BMP2, BMP4, BMP6 and BMP7 (10 ng/mL) selected for these experiments were considered optimal based on their modulatory effects on androstenedione secretion observed in our previous studies on bovine TC (Glister et al. 2005 (link)(Glister et al. , 2010 (link)(Glister et al. , 2011)) (link). Each BMP-binding protein was tested at three different concentrations (50, 250, 1250 ng/mL) for its ability to antagonize BMP-induced suppression of androstenedione secretion by LH-stimulated cells. Co-treatments were prepared 30-40 min before addition to cells by mixing appropriate concentrations of BMP and BMP-binding protein. A further experiment examined the effect of 24, 48 and 96 h exposure to BMP6 (10 ng/mL) alone on the relative abundance of CHRD, GREM1, NOG, FST, BMP2, BMP4, BMP6, BMP7 and SMAD6 mRNA.
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