hESC were plated on collagen I (Corning)–coated 6-well plates (Corning), 24-well plates (Corning), or glass coverslips (15 mm). Cells were cultured in EpiLife medium until 70 to 80% confluence. The culture medium was replaced by the chemical induction medium consisting of DMEM/F12 (Invitrogen, #10565018), 0.5% N2 (Gibco, #17502048), 0.5% B27 (Gibco, #17504044), and 1% P/S, supplemented with 5 μM Repsox (MedChem Express, #HY-13012), 3 μM CHIR99021 (MedChem Express, #HY-10182), bFGF (10 ng/ml; PeproTech, #100-18B-50), and BMP4 (10 ng/ml; PeproTech, #120-05ET-10). The control group was treated with the basal medium containing 1% dimethyl sulfoxide in the absence of small molecules and growth factors. The chemical induction medium was refreshed every 2 days. After 6 to 8 days of induction, hESC were induced into GCs, and the induced GCs were maintained and passaged in the chemical induction medium for the following use. Other small molecules used in the study included 3 μM Kenpaullone (MedChem Express, #HY-12302), 5 μM A83-01 (MedChem Express, #HY-10432), and 5 μM SB431542 (Selleck, #S1067), and 5 μM DAPT (MedChem Express, HY-13027 ).
A83 01
Manufactured by MedChemExpress
Sourced in United Kingdom, China, United States
A83-01 is a small molecule inhibitor. It functions as a selective inhibitor of the transforming growth factor beta (TGF-β) type I receptor ALK5.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by MedChemExpress (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Y 27632, by MedChemExpress (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Chir99021, by Selleck Chemicals (2 mentions)
Fibroblast growth factor basic bfgf, by Thermo Fisher Scientific (2 mentions)
Low glucose dmem, by Thermo Fisher Scientific (2 mentions)
Y 27632, by Selleck Chemicals (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Repsox, by MedChemExpress (1 mentions)
Sb431542, by Selleck Chemicals (1 mentions)
24 well plate, by Corning (1 mentions)
Bt 549, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Pci 34051, by MedChemExpress (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
11 protocols using a83 01
1
Directed Differentiation of hESCs into Germ Cells
2
Breast Cancer Cell Line Culture Protocols
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The breast cancer cell lines 4T1, MDA-MB-231, BT549 and HEK 293 human kidney cells were obtained from the American Type Culture Collection (ATCC®). 4T1, MDA-MB-231 and HEK 293 cells were cultured in high glucose DMEM (Gibco®) supplemented with 10% FBS (Gibco®). BT549 cells were cultured in RPMI-1640 (Gibco®) supplemented with 10% FBS. All cells were maintained at 37°C in a humidified 5% CO2 atmosphere. The ALK5 inhibitor A8301, HDAC8 inhibitor PCI-34051 and paclitaxel were obtained from MedChemExpress (MCE®). Other HDAC inhibitors mentioned in manuscript were from Selleck.
3
Liver Organoid Differentiation Protocol
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Liver organoids were cultured for 7 days in expansion medium (EM) supplemented with bone morphogenetic protein 7 (BMP7, 25 ng/ml) (MedChemExpress, USA). Medium was then changed into the differentiating medium (DM) which consisted of AdDMEM/F12 supplemented with 1% of both N2 and B27, 50 ng/ml EGF (Peprotech, UK), 10 nM gastrin (Sigma, USA), 25 ng/ml HGF (Peprotech, UK), 100 ng/ml FGF19 (Peprotech, UK), 500 nM A83-01 (MedChemExpress, USA), 10 μM DAPT (MedChemExpress, USA), 25 ng/ml BMP7 (MedChemExpress, USA), and 30 μM dexamethasone (MedChemExpress, USA). Differentiation medium was changed every 2–3 days for 13–15 days of culture. Functional analysis was performed in the collected supernatant (24 h after the last medium change) or in whole organoids.
4
Expansion and Characterization of Expanded ACY-Treated MSCs
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In an ACY (-) group, MSCs were cultured as previously described (Henrionnet et al., 2017 (link); Samal et al., 2021 (link)). In brief, the culture medium contained low glucose DMEM (Gibco) supplemented with FBS (10%, Hyclone), fibroblast growth factor-basic (bFGF, 1 ng/ml, PeproTech), and penicillin/streptomycin (1%, Gibco). In ACY (+) group, the basal medium for culturing MSCs consisted of low glucose DMEM, FBS, bFGF, penicillin/streptomycin, and ACY cocktails consisted of A-83–01 (MedChemExpress), CHIR99021 (Selleck), and Y-27632 (Selleck). The medium was changed 1 day after seeding and every 2 days thereafter. There were 2 μM A-83–01, 3 μM CHIR99021, and 2 μM Y-27632 chosen as the optimal concentration in the ACY (+) group. When cells were approximately 90% confluent, they would be passaged with a split ratio of 1:3.
MSCs at passage 2 (P2) were seeded, without (ACY-) or treated (ACY+) with A-83–01, CHIR99021, and Y-27632, and were continuously cultured to passage 10 (P10;Supplementary Figure S1 ). From passage 5 (P5) to P10, the MSCs were used in subsequent analysis.
MSCs at passage 2 (P2) were seeded, without (ACY-) or treated (ACY+) with A-83–01, CHIR99021, and Y-27632, and were continuously cultured to passage 10 (P10;
5
Blastoid Induction from Naive hPSCs
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Naive hPSCs cultured on MEFs in PXGL medium were pre-treated with PRC2 inhibitor (1 μM UNC1999) for 4 d before blastoid induction. Blastoids were induced19 as follows. Naive hPSCs cultured in untreated or pre-treated conditions were harvested using accutase (Biozym). The cells were resuspended in PXGL medium supplemented with 10 µM Y-27632 (MedChemExpress), seeded onto gelatin-coated plates and incubated at 37 °C for 70 min to deplete the MEFs. The unattached cells were collected, pelleted through centrifugation and resuspended in N2B27 medium containing 10 µM Y-27632 with or without PRC2 inhibitor (aggregation medium), after which 30,000 cells were seeded onto an array of 200-µm microwells inserted into a well of a 96-well plate. Note that microwell arrays comprising microwells were imprinted into 96-well plates77 (link),78 (link). After 24 h, the aggregation medium was replaced with N2B27 medium supplemented with 1 μM PD0325901, 1 μM A83-01 (MedChemExpress, HY-10432), 500 nM 1-oleoyl lysophosphatidic acid sodium salt (Tocris, 3854), 10 ng ml−1 hLIF and 10 µM Y-27632, with DMSO for control or 1 μM UNC1999 for pre-treated samples. The medium was refreshed every 24 h.
6
Liver Progenitor Cell Generation Protocol
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The generation of liver progenitor cells were basically according to previously published protocol17 (link). Concisely, isolated primary mouse hepatocytes were seeded on plates coated with collagen type I (Sigma) at 2 × 104 cells/cm2. The cells were cultured in DMEM supplemented with 10% FBS for 6 h and then the medium changed to SHM medium (DMEM/F12 containing 2.4 g/L NaHCO3 and L-glutamine (Gibco) supplemented with 5 mM HEPES (Solarbio), 30 mg/L L-proline (Alfa Aesar), 0.05% BSA (Solarbio), 10 ng/ml epidermal growth factor (PeproTech), insulin-transferrin-serine (ITS) (Sigma-Aldrich), 10−7 M dexamethasone (Dex) (Sigma-Aldrich), 10 mM nicotinamide (Solarbio), 1 mM ascorbic acid-2 phosphate (Wako) and 1× antibiotic/antimycotic solution (Solarbio)) supplemented with YAC (10 mM Y-27632 (Medchemexpress), 0.5 mM A-83-01 (Medchemexpress), 3 mM CHIR99021 (Medchemexpress)). Cells were cultured for 14 days to generate liver progenitor cells and the medium was changed every other day thereafter.
7
Isolation and Culture of Human MSCs
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Following the Clinical Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University and written informed patient consent for tissue collection, MSCs were isolated following the protocol described in the previous study (23 (link)). In brief, fat tissues were isolated via liposuction from healthy patients defined as no history of malignancy or autoimmune deficiency. The fat tissues were shredded and digested with 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes at 37°C with gentle agitation. The cells were collected and cultured with MSC medium containing low glucose DMEM (Gibco) supplemented with FBS (10%, Gibco), fibroblast growth factor-basic (bFGF, 1 ng/ml, PeproTech) and penicillin/streptomycin (1%, Gibco).
When MSCs were approximately 90% confluent, they would be passaged with a split ratio of 1:3. MSCs of passage 2 were suspended in MSC medium with or without combinations of the following three small molecules, 2μM A-83-01 (MedChemExpress), 3μM CHIR99021 (Selleck), and 2μM Y-27632 (Selleck), and seeded on plates. The medium was changed every 2 days. From passage 5 to passage 7, the MSCs were cultured in MSC medium and used in subsequent analysis.
When MSCs were approximately 90% confluent, they would be passaged with a split ratio of 1:3. MSCs of passage 2 were suspended in MSC medium with or without combinations of the following three small molecules, 2μM A-83-01 (MedChemExpress), 3μM CHIR99021 (Selleck), and 2μM Y-27632 (Selleck), and seeded on plates. The medium was changed every 2 days. From passage 5 to passage 7, the MSCs were cultured in MSC medium and used in subsequent analysis.
8
Cardiomyocyte Hypoxia Model using GiWi
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Cardiomyocyte differentiation was conducted in a growth factor and serum-free system following the Gsk3 inhibitor and Wnt inhibitor (GiWi) protocol [14 (link)], in which the canonical Wnt signaling pathway was manipulated using a Gi and Wi.
For hypoxia treatment, iPSC-CMs after indicating culture periods were subjected to hypoxic culture (1% O2, 5% CO2, 94% N2) for 24 h and reoxygenation for 6 h. Then, the cells were subjected to cell counting kit (CCK)-8 assays for cell viability and lactate dehydrogenase (LDH) cytotoxicity assays. Recombinant human Hapln1 (rhHapln1) protein was obtained from Sino Biological (10323-H08H; Beijing, China). Recombinant mouse HAPLN1 (rmHAPLN1), human growth differentiation factor 11 (GDF11), and human Nodal growth differentiation factor (NODAL) protein were obtained from CUSABIO (CSB-EP010130MO, CSB-EP009344HU, and CSB-EP850428HU; Wuhan, China).
A-83-01, a potent inhibitor of the transforming growth factor (TGF)-β type I receptor ALK5, ALK4, and ALK7 kinase and GDF11 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). GDF11 neutralizing antibody was purchased from Creative BioLabs (CAT#: NEUT-859CQ; Shirley, NY, USA).
For hypoxia treatment, iPSC-CMs after indicating culture periods were subjected to hypoxic culture (1% O2, 5% CO2, 94% N2) for 24 h and reoxygenation for 6 h. Then, the cells were subjected to cell counting kit (CCK)-8 assays for cell viability and lactate dehydrogenase (LDH) cytotoxicity assays. Recombinant human Hapln1 (rhHapln1) protein was obtained from Sino Biological (10323-H08H; Beijing, China). Recombinant mouse HAPLN1 (rmHAPLN1), human growth differentiation factor 11 (GDF11), and human Nodal growth differentiation factor (NODAL) protein were obtained from CUSABIO (CSB-EP010130MO, CSB-EP009344HU, and CSB-EP850428HU; Wuhan, China).
A-83-01, a potent inhibitor of the transforming growth factor (TGF)-β type I receptor ALK5, ALK4, and ALK7 kinase and GDF11 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). GDF11 neutralizing antibody was purchased from Creative BioLabs (CAT#: NEUT-859CQ; Shirley, NY, USA).
9
Establishing 3D Organoid Culture of Triple-Negative Breast Cancer
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TNBC tissues were cut into small pieces and mechanically homogenised. The homogenised tissue was digested with trypsin on ice. After centrifugation, single‐cell suspensions were added dropwise with cold 3D Matrigel matrix (Corning, 354348) to six‐well culture plates, which were kept inverted in a cell incubator for 30 min before organoid‐specific medium was added. Organoids were cultured with media containing 1% GlutaMax (Thermo Fish, 35050061), 100 μg/mL Primocin (InvivoGen, 26‐69‐PM), .5 μg/mL hydrocortisone (MedChemExpress, HY‐N0583), 10 μM Y27632 (Sigma, Y0503), .2 nM Wnt3a (StemRD, W3a‐H‐025), 250 ng/mL R‐spondin1 (PeproTech, 120‐38), 100 ng/mL Noggin (PeproTech, 120‐10D), 1× B27 + Vitamin A (Invitrogen, 17504‐044), 10 mM HEPES (Thermo Fish, 15630080), 20 ng/mL Fibroblast Growth Factor‐10 (FGF‐10) (Peprotech, 100‐26), 5 ng/mL Epidermal Growth Factor (EGF) (Peprotech, GMP100‐15), 10 mM nicotinamide (Selleckchem, S1899), 100 nM β‐estradiol (Sigma, E2758), 10 μM Forskolin (MedChemExpress, HY‐15371), 5 nM Heregulin B1 (ProSpec, CYT‐733), .5 μM A83‐01 (MedChemExpress, HY‐10432), .5 μM SB202190 (Selleckchem, S10077), 1.25 mM N‐acetylcysteine (Sigma, A0737) and Dulbecco's Modified Eagle Medium (DMEM).
10
Culture of Human Trophoblast Stem Cells
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The culture of human trophoblast was performed as described previously47 (link). Briefly, the plate was coated with 5 μg ml−1 Collagen I (Corning) at 37 °C at least for 1 h. hTSCs were cultured in hTSCs medium (DMEM/F12 (Gibco) supplemented with 0.1 mM 2-mercaptoethanol (Gibco), 0.2% FBS (Gibco), 0.5% Penicillin–Streptomycin (Gibco), 0.3% BSA (Sigma-Aldrich), 1% ITS-X supplement (Gibco), 1.5 μg ml−1l -ascorbic acid (Sigma-Aldrich), 50 ng ml−1 Epidermal Growth Factor (EGF, MedChemExpress), 2 μM CHIR99021 (MedChemExpress), 0.5 μM A83-01 (MedChemExpress), 1 μM SB431542(MedChemExpress), 0.8 mM valproic acid (VPA, Wako) and 5 μM Y27632 (MedChemExpress)). hTSCs were dissociated with TrypLE (Gibco) for 8 min at 37 °C, and the cells were passaged to a new Collagen I-coated plate. hTSCs were routinely passaged every 4–5 d at a 1:4–1:6 ratio.
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