All materials were obtained from ThermoFisher Scientific unless otherwise noted. Apyrase and 2-MeSADP, were purchased from Sigma (St. Louis, MO). PAR 4 peptide AYPGKF-NH2 and PAR 1 peptide SLFLLRN-NH2 were synthesized by GenScript (Piscataway, NJ). Anti-Akt (Ser473) cat#4060 was obtained from Cell Signaling (Danvers, MA). Antibody to total Akt cat#TA504230 was purchased from Origene (Rockville, MD). Blocking buffer, infrared dye-labeled goat anti-mouse (926-68020) and anti-rabbit antibodies (926-32211) were purchased from LI-COR (Lincoln, NE). PI103 was purchased from Tocris Bioscience (Minneapoli, MN)
Pi 103
Manufactured by Bio-Techne
Sourced in United Kingdom
PI-103 is a potent and selective inhibitor of the PI3-kinase pathway. It targets the p110α, p110β, p110δ, and p110γ isoforms of PI3-kinase with high potency. PI-103 can be used in research applications to study the role of the PI3-kinase pathway in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Activin a, by R&D Systems (3 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Bio-Techne (2 mentions)
Ldn193189, by Bio-Techne (2 mentions)
Hyclone fbs, by Bio-Techne (2 mentions)
Hyclone fbs, by GE Healthcare (2 mentions)
Y 27632, by Bio-Techne (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Torin1, by Bio-Techne (2 mentions)
Apyrase, by Merck Group (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Antibody to total akt cat ta504230, by OriGene (1 mentions)
Infrared dye labeled goat anti mouse 926 68020, by LI COR (1 mentions)
Anti rabbit antibodies 926 32211, by LI COR (1 mentions)
2 mesadp, by Merck Group (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Ldn 193189, by R&D Systems (1 mentions)
A83 01, by Bio-Techne (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Forskolin, by Bio-Techne (1 mentions)
11 protocols using pi 103
1
Kinase Inhibition Signaling Assay
2
Directed Differentiation of Endoderm
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Endodermal differentiation was performed as previously described [38 (link)]. Pluripotent human stem cells were grown in the absence of MEF for four passages in mTeSR1 (StemCell Technologies, 85850) and seeded on Geltrex (invitrogen, A1413202). After 1–2 days of recovery in mTeSR1, hESC were washed with F12 (Gibco, 21765–029) and then were treated for 24 hours with Activin A (100 ng/mL, R&D Systems, 338-AC-010), CHIR99021 (2 μM, Stemgent, 04–0004), and PI-103 (50 nM, Tocris, 2930) in CDM2 to specify APS. Afterwards, cells were washed (F12), then treated for 48 hours with Activin A (100 ng/mL) and LDN-193189/DM3189 (250 nM, Stemgent, 04–0074) in CDM2 to generate DE by day 3. Day 3 DE was patterned into AFG, PFG, or MHG by 4 days of continued differentiation in CDM2. DE was washed (F12), then differentiated as follows: AFG, A-83-01 (1 μM, Tocris, 2939) and LDN-193189 (250 nM, Stemgent, 04–0074); PFG, RA (2 μM, Sigma, R2625) and LDN-193189 (250 nM); MHG, BMP4 (10 ng/mL, R&D Systems, 314-BP-010), CHIR99021 (3 μM, Stemgent, 04–0004), and FGF2 (100 ng/mL, Peprotech, 100-18B), yielding day 7 anteroposterior domains. Media was refreshed every 24 hours for each differentiation step.
3
Directed Differentiation of hESCs into Endoderm
4
Characterization of Cellular Signaling Pathways
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Forskolin, MK-2206 and PI-103 were from Tocris (Avon, UK). Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence reagents were purchased from GE Healthcare (Piscataway, NJ, USA). [γ32P]-labelled ATP was from Perkin Elmer (MA, USA). Precision Plus protein markers and SsoFast EvaGreen Supermix were from Bio-Rad (Hertfordshire, UK). Insulin-like growth factor 1 (IGF-1) was from Cell Signaling Technology (Hertfordshire, UK). Protease inhibitor cocktail tablets were purchased from Roche (Lewes, UK). Anti-HA-agarose, anti-FLAG-agarose, collagenase (from clostridium histolyticum type IV, Cat no. C5138), triiodothyronine, Bt2-cAMP and lactate were from Sigma-Aldrich (Poole, UK). Infinity glucose assay kit was from Thermo Scientific (Essex, UK). Insulin and glucagon were from Novo Nordisk (Sussex, UK). Cell culture media and reagents including Dulbecco’s modified eagle medium (DMEM) and M199 with glutamax were from Life Technologies (Paisley, UK). All other chemicals unless specified were obtained from Sigma-Aldrich.
5
PI3K Inhibition Assay Protocol
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Naringenin (PS010691, >99.0%), tangeretin (PS010637, >98.0%), luteolin (PS010346, >99.0%), hesperetin (PS000219, >98.0%), and auraptene (PS010582, >98.0%) were purchased from Chengdu Push Bio-technology Co., Ltd. (Sichuan, China). PI3Kα (p110α/p85α) was purchased from Invitrogen (Carlsbad, California, USA). PI3Kβ (p110β/p85α) was purchased from Eurofins (Brussels, Belgium). PI3Kγ (p120γ) and PI3Kδ (p110δ/p85α) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ADP-Glo Kinase Assay Kit was purchased from Promega (Madison, WI, USA). PIP2 was purchased from Life Technologies (Carlsbad, California, USA). Dimethyl sulfoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PI103 (Lot number: 3 A/122414), a multi-targeted PI3K inhibitor, was purchased from Tocris Bioscience (Bristol, UK).
6
Cell Culture and Pharmacological Interventions
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ELT3 cells (Eker rat uterine leiomyoma-derived cells; Howe et al., 1995a (link),b (link)) and LAM patient–derived 621–101, 621–102, and 621–103 cells were cultured in IIA complete medium. Tsc2−/−p53−/− MEFs, HEK293, HeLa, U2OS, and OVARC5 cells were cultured in DMEM supplemented with 10% FBS. 17-β-estradiol (10 nM; Sigma-Aldrich), rapamycin (20 nM; Biomol), Torin 1 (250 nM; Tocris), LY294002 (20 µM; Cell Signaling Technology), NS398 (50 µM; Cayman Chemical), Sulindac (50 µM; Cayman Chemical), aspirin (450 µM; Sigma-Aldrich), Celecoxib (Novartis), PI-103 (5 µM; Tocris), and AktVIII (5 µM; Millipore), and Wortmannin (1 µM; Cell Signaling Technology) were used as indicated. 15-epi-LXA4 (10–500 nM) was obtained from Millipore. The integrity of 15-epi-LXA4 was determined by UV-Vis spectrometry and HPLC. Because of the volatile nature of this product, stock solutions were stored at −80°C.
7
Inhibitor-Mediated Modulation of Signaling Pathways
8
Chemotaxis Assay for Cell Migration
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CCL21 and CXCL12 were from Peprotech, and CXCL13 was purchased from R&D systems. Chemotaxis assays were carried out using Transwell chambers (5 µm pore size; CoStar) adding 100 µl cell suspension (5 x 106 cells/ml) in complete medium (RPMI/10% FCS/standard supplements) to the top chamber and indicated amounts of chemokine in the bottom chamber. After 2 h at 37°C, 7% CO2, the percentage of migrated cells was calculated by flow cytometry after comparing with a precalibrated bead standard (Sigma-Aldrich) and correcting for variations in input concentrations. The DOCK2 inhibitor CPYPP (Selleck) was used at 40 µM throughout the chemotaxis assay (15 (link)). The isoform-specific PI3K inhibitors TGX221 (0.1 µM final conc.; Tocris), PI-103 (1 µM; Tocris), AS604850 (1 µM; Selleck),and IC-87114 (0.5 µM; Selleck) were present throughout the chemotaxis assay.
9
Definitive Endoderm Differentiation Protocol
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Definitive endoderm was induced according to Loh et al. (2014) (link). Cells were cultured in CDM2 basal medium that was supplemented with 100 ng/ml activin A (produced in house), 100 nM PI-103 (Tocris Bio-Techne, 2930), 3 µM CHIR99021, 10 ng/ml FGF2 (produced in house), 3 ng/ml BMP4 (PeproTech, 120-05ET), 10 µg/ml heparin (Sigma-Aldrich, H3149) for one day. For the next 2 days the following supplements were applied: 100 ng/ml activin A, 100 nM PI-103, 20 ng/ml FGF2, 250 nM LDN193189, 10 µg/ml heparin. Further induction of foregut progenitors was performed according to Rezania et al. (2014) (link) with analysis at the S4 stage.
10
Directed Differentiation of hESCs into Endoderm
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H1-AAVS1-TetOn-dCas9-KRAB hESCs were split to single cells with
TrypLE Express (Thermo, 12604). Cells were resuspended in mTeSR1
supplemented with 10 μM Y27632 (Tocris, 1254) and 500 ng/mL
doxycycline. 2 × 106 cells were plated into each well of a
6-well plate pre-coated with Growth Factor Reduced Matrigel (Corning,
356231). On Day 1, cells were fed with mTeSR1. On Day 2, cells were fed with
RPMI1640 (Thermo, 21870) supplemented with 0.2% Hyclone FBS (GE Healthcare,
SH30070.03), 100 ng/mL Activin A (R&D Systems, 338-AC-01M), 3 μM
CHIR 99021 (Tocris, 4423), and 50 nM PI 103 (Tocris, 2930). On Days 3 and 4,
cells were fed with RPMI1640 supplemented with 0.2% Hyclone FBS, 100 ng/mL
Activin A, and 250 nM LDN-193189 (Tocris, 6053). Media was changed every 24
hours. For perturbation experiments and CRISPRi screening, the media was
supplemented with 500 ng/mL doxycycline daily.
TrypLE Express (Thermo, 12604). Cells were resuspended in mTeSR1
supplemented with 10 μM Y27632 (Tocris, 1254) and 500 ng/mL
doxycycline. 2 × 106 cells were plated into each well of a
6-well plate pre-coated with Growth Factor Reduced Matrigel (Corning,
356231). On Day 1, cells were fed with mTeSR1. On Day 2, cells were fed with
RPMI1640 (Thermo, 21870) supplemented with 0.2% Hyclone FBS (GE Healthcare,
SH30070.03), 100 ng/mL Activin A (R&D Systems, 338-AC-01M), 3 μM
CHIR 99021 (Tocris, 4423), and 50 nM PI 103 (Tocris, 2930). On Days 3 and 4,
cells were fed with RPMI1640 supplemented with 0.2% Hyclone FBS, 100 ng/mL
Activin A, and 250 nM LDN-193189 (Tocris, 6053). Media was changed every 24
hours. For perturbation experiments and CRISPRi screening, the media was
supplemented with 500 ng/mL doxycycline daily.
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