The University of Arizona Institutional Animal Care and Use Committee approved all animal protocols. The C57BL/6J mice were fed on laboratory food and tap water ad libitum in a regular 12 hours dark/light cycle. Postnatal day (P) 3 newborn pups were injected daily with DAPT (Sigma, 50μg/g), Jag1 peptide (AnaSpec, 50 μg/g), Y-27632 (Sigma, 10μg/g), the combination of DAPT and Y-27632, or the combination of Jag1 and Y27632 daily for 4 days. The control pups were injected with 1× PBS. The mouse pups were humanely sacrificed at P7. The eyelids were cut and the eyes were enucleated using a pair of curved forceps to pinch the optic nerve and ocular muscles. The eyes were transferred to a petri dish containing 4% paraformaldehyde in artificial cerebrospinal fluid solution for 5-10 minutes. The eyes were transferred to a petri dish containing Dulbecco’s phosphate-buffered saline placed under a dissection microscope to dissect the retina.
Y 27632
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Canada, Australia, Switzerland, Israel, China, Morocco
Y-27632 is a selective and potent Rho-associated protein kinase (ROCK) inhibitor. It functions by inhibiting the activity of ROCK, a key enzyme involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Blebbistatin, by Merck Group (80 mentions)
Glutamax, by Thermo Fisher Scientific (59 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (50 mentions)
Matrigel, by Corning (47 mentions)
N acetylcysteine, by Merck Group (41 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (41 mentions)
Nicotinamide, by Merck Group (39 mentions)
Dmem f12, by Thermo Fisher Scientific (38 mentions)
A83 01, by Bio-Techne (35 mentions)
Hepes, by Thermo Fisher Scientific (33 mentions)
Cytochalasin d, by Merck Group (32 mentions)
Matrigel, by BD (29 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (29 mentions)
Nocodazole, by Merck Group (26 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (21 mentions)
Tryple express, by Thermo Fisher Scientific (20 mentions)
Ck 666, by Merck Group (20 mentions)
L glutamine, by Thermo Fisher Scientific (18 mentions)
Noggin, by Thermo Fisher Scientific (18 mentions)
B27 supplement, by Thermo Fisher Scientific (18 mentions)
775 protocols using y 27632
1
Neonatal Retinal Development Modulation
2
Genome Editing of Patient-Derived iPSCs
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Patient-specific iPSCs on MatrigelTM coated dishes were used for transfection. After incubation with 10 μM Y27632 (Sigma-Aldrich) for 2 hours, cells were detached with trypLETM Select (Life Technologies) and counted. Then the cells were resuspended with 100 μL Human Stem Cell Nucleofector Kit 2 (Lonza) and nucleofected using Nucleofector II (Lonza) set at program B016. For each experiment, 5 μg of linearized donor vector was used with or without the 5 μg of the TALENs. The transfected cells were plated on PMEF-NL (Millipore) feeders in ES medium containing 10 μM Y27632. G418 selection (50 μg/ml, Calbiochem) was started 48–72 hours after transfection. About two weeks later, resistant clones were picked and expanded for further analysis.
For excision of PGK-Neo, cells on matrigel were detached with trypLETM Select and pelleted. Then the cells were resuspended with 100 μL Human Stem Cell Nucleofector Kit 2 with 5 μg pCAG-cre (addgene) and nucleofected using Nucleofector II set at program B016. Ten thousand of nucleofected cells were seeded on 6 cm dish with feeders in ES medium containing 10 μM Y27632. After two weeks of continuous culture without drug selection, single clones were picked and expanded for further analysis.
For excision of PGK-Neo, cells on matrigel were detached with trypLETM Select and pelleted. Then the cells were resuspended with 100 μL Human Stem Cell Nucleofector Kit 2 with 5 μg pCAG-cre (addgene) and nucleofected using Nucleofector II set at program B016. Ten thousand of nucleofected cells were seeded on 6 cm dish with feeders in ES medium containing 10 μM Y27632. After two weeks of continuous culture without drug selection, single clones were picked and expanded for further analysis.
3
Rho-kinase Inhibitor Treatment Assay
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A pharmacological inhibitor of the Rho-associated kinase (ROCK) pathway, Y-27632 (Sigma-Aldrich), was diluted in stock dimethyl sulfoxide (DMSO) and was then added to the culture medium for final drug concentrations of 20 μM, 10 μM, and 5 μM. Cells were plated for 23 hours and then incubated for 1 hour in Y-27632 before fixation. Control experiments were performed with drug-free medium containing DMSO at the same concentration as in the drug medium to assure that DMSO was not affecting the cells.
4
Culturing Human Intestinal Epithelial Cells
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Adult secretor positive jejunal HIE cultures (J2 cell line) were grown as undifferentiated 3D cultures as described previously with minor modifications (68 (link)). Briefly, HIEs were recovered from liquid nitrogen (LN2), suspended in 20 μL of Matrigel (Corning, cat. no. 354234), plated in a single well of a 24-well plate, and grown as 3D cultures in 500 μL of IntestiCult Human Organoid Growth Medium (OGM; STEMCELL Technologies, cat. no. 06010) supplemented with 10 μM Y-27632 (Sigma-Aldrich, cat. no. Y0503). Medium was refreshed every other day. Single-cell suspensions were obtained upon treatment of the HIE with 0.05% Trypsin/0.5mM EDTA (Invitrogen, cat. no. 25300054), resuspended in OGM with Y-27632, and plated as undifferentiated monolayers in collagen IV (Sigma-Aldrich, cat. no. CC076) pre-coated 96-well plates. After 24 h, culture medium was replaced with differentiation medium to induce cell differentiation. Differentiation medium compromises equal volumes of complete medium without growth factors (CMGF− advanced DMEM/F12 supplemented with 1% GlutaMAX, 1% penicillin/streptomycin, 1% 1 M HEPES) and Intesticult OGM Basal media. Cells were differentiated for 4 days. Medium was refreshed every other day.
5
Mesenchymal Stem Cell Induction from hESCs
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hESC lines (H1 hESCs, WiCell Research Institute) were cultured as we previously reported [13 (link), 14 (link)]. Briefly, the cells were seeded on 12-well plate (Corning) coated with matrigel (BD Bioscience) in mTeSR1 medium (Stem Cell Technologies) at 5% CO2 and 37 °C. For MSC induction, hESCs were split into single cells with Accutase (Gibco), then seeded in mTeSR1 medium with Y27632 (10 nM) (Sigma) addition on a growth factor reduced gel (GFR, Stem Cell Technologies)-coated 6-well plate at a density of 1.3 × 104 cells/mL. After 2 days, the spent medium was replaced by DMEM/F12 basal medium (Hyclone) containing 5% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco), 1% l -glutamine (Gibco), and 10 nM small molecule (Targetmol) from day 0 to day 7; then, cells were transferred into an adherent culture plate in DMEM/F-12 medium supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco), 1% l -glutamine (Gibco), and 5 nM Y27632 (Sigma). Cells were cultured for another 7 days, and media were changed every 2 days in the entire process.
6
Directed Differentiation of hiPSC-bKs
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On day 25 of differentiation, hiPSC-bKs were collected with Accumax (Gibco, Life Technologies, Carlsbad, CA, USA) and plated onto collagen I-coated dishes (100 µg/mL, Advanced Biomatrix) in CnT-07 media (CellnTec, Bern, Switzerland) supplemented with 10 µM Y-27632 (Sigma-Aldrich, Saint Louis, MO, USA). A rapid attachment step of approx. 20 min was performed to allow attachment of only hiPSC-bKs. Cells were fed every other day until confluent when they were used for downstream analysis. For assessing terminal differentiation ability of hiPSC-bKs and control hKs, cells were changed to CnT-PR-D media (CellnTec, Bern, Switzerland) without Y-27632 24h before confluency and, the next day, 1.2 mM CaCl2 (Sigma-Aldrich, Saint Louis, MO, USA) was added to the differentiation media. Samples were taken for analysis after 4 and 8 days.
7
Differentiation of iPSCs into Endothelial Cells
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Differentiation was initiated with B2M−/−CIITA−/− rhCD47 tg hiPSC at 60% confluency, and medium was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 µM CHIR-99021 (Selleckchem). On day 2, the medium was changed to reduced medium: RPMI-1640 containing 2% B-27 minus insulin (Gibco) and 2 µM CHIR-99021 (Selleckchem). From days 4–7, cells were cultured in RPMI-1640 EC medium of RPMI-1640 containing 2% B-27 minus insulin plus 50 ng/ml human VEGF, 10 ng/ml human FGFb; R&D Systems), 10 µM Y-27632 (Sigma-Aldrich), and 1 µM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible from day 7 and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell) plus 10% FCS hi (Gibco), 1% pen/strep, 25 ng/ml VEGF, 2 ng/ml FGFb, 10 µM Y-27632 (Sigma-Aldrich), and 1 µM SB 431542 (Sigma-Aldrich). The differentiation process was completed after 14 d and undifferentiated cells detached during the differentiation process. TrypLE Express (Gibco) was used for passaging the cells 1:3 every 3–4 d.
8
Efficient Generation of iPSC Clones
9
Corneal Injury Repair: PF562271 and Y27632
10
TGFβ1-Induced Cellular Responses
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Where indicated, AECs 24 h post‐TGFβ1 application or MCs were treated with TGFβ1 inhibitor (ALK5i, Boehringer Ingelheim, EX76980, 0.2 µM), ROCK inhibitor (Y27632, Sigma‐Aldrich, 10 µM), EZH2 inhibitor (GSK126, 10 nM), TAK1 inhibitor (5Z‐7‐Oxozeaenol; 5‐OZ, Sigma‐Aldrich, 1 µM), POL2‐S2p inhibitor (5,6‐Dichlorobenzimidazole 1‐β‐D‐ribofuranoside; DRB, Sigma‐Aldrich, 100 µM) and TGFβ1 (R&D, 240‐B, 1 ng/ml). The vehicle dimethyl sulphoxide (DMSO) was used as control for ALK5, DRB, GSK126, Y27632 and 5‐OZ treatments, 4 mM HCl (Sigma‐Aldrich) containing 1 mg/ml BSA (Sigma‐Aldrich) was used as control for TGFβ1 treatment.
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