Rock inhibitor y 27632

Manufactured by STEMCELL

Sourced in Canada, United States, United Kingdom

ROCK inhibitor Y-27632 is a chemical compound that inhibits the activity of Rho-associated protein kinase (ROCK), an enzyme involved in various cellular processes. It functions by blocking the catalytic domain of ROCK, thereby modulating the downstream signaling pathways regulated by this enzyme.

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Lab products found in correlation

Accutase, by STEMCELL (13 mentions) Mtesr1 medium, by STEMCELL (9 mentions) Mtesr1, by STEMCELL (6 mentions) Glutamax, by Thermo Fisher Scientific (6 mentions) Matrigel, by Corning (5 mentions) Matrigel coated, by Corning (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Matrigel matrix, by Corning (4 mentions) Essential 8 medium, by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Opti mem, by Thermo Fisher Scientific (4 mentions) Facsaria cell sorter, by BD (3 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions) Matrigel coated plate, by Corning (3 mentions) Relesr, by STEMCELL (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Gentle cell dissociation reagent (gcdr), by STEMCELL (3 mentions) Activin a, by R&D Systems (3 mentions) Pou5f1 egfp h1 hesc line, by WiCell (3 mentions) Matrigel, by BD (3 mentions)

75 protocols using rock inhibitor y 27632

Cultivation and Characterization of Human Corneal Endothelial Cells

Cited 3 times

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Our experiment on cultivated HCECs was composed of two main parts (ELISA in part one and cell counting, morphology, and immunocytochemistry [ICC] in the second part). Each part was performed in triplicate. For each run, the DMs from six different cornea donors were pooled and incubated in 3.5 mg/ml collagenase A (Roche, USA) at 37°C for 50 min and then centrifuged at 300 g for 5 min. Finally, the pooled cells were cultured in DMEM: F12 supplemented with 20% fetal bovine serum (FBS; Gibco), 120 mg/ml penicillin (Sigma, Germany), and 220 mg/ml streptomycin (Sigma, Germany) on a 24-well plate coated with 20 mg/ml of fibronectin (Sigma, USA) at 37°C and 5% CO

_{2}

. All experiments were performed when the cells were in P1. For this purpose, after two weeks of cell isolation and culture, the cells were passaged by trypsin/EDTA and seeded on a 24-well plate at a seeding density of 1

\times

^{4}

cells per well with DMEM: F12 culture medium supplemented with three different treatment groups including 20% HPL, 10 μM Y-27632 ROCK inhibitor (STEMCELL Technologies, USA), combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. The concentration of Y-27632 ROCK inhibitor was designated based on the previous studies.^{[18 (link),27 (link),28 (link)]}

Mishan M.A., Balagholi S., Chamani T., Feizi S., Soheili Z.S, & Kanavi M.R. (2021). Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor. Journal of Ophthalmic & Vision Research, 16(3), 349-356.

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Prostate Single-Cell Suspension Immunophenotyping

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Mouse prostate single cell suspension was blocked with the Fc blocker (CD16/32 antibody from eBioscience) for 40 min at room temperature. Staining antibodies were diluted in 4% FBS buffer containing Y-27632 ROCK inhibitor (STEMCELL Technologies, #72302) and applied to the prostate single cell suspension for 40 min at room temperature. Flow cytometry analysis or sorting was conducted using BD Accuri C6 or FACSAria III flow cytometer. Antibodies used in the study are listed in Supplementary Table 2. Propidium Iodide (PI, Invitrogen) was added to sample before sorting to gate viable cells.

Wang X., Xu H., Cheng C., Ji Z., Zhao H., Sheng Y., Li X., Wang J., Shu Y., He Y., Fan L., Dong B., Xue W., Wai Chua C., Wu D., Gao W.Q, & He Zhu H. (2020). Identification of a Zeb1 expressing basal stem cell subpopulation in the prostate. Nature Communications, 11, 706.

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Endothelial Barrier Regulation Assay

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Endothelial barrier function was assessed by measuring trans-endothelial electrical resistance (TEER) using the electric cell-substrate impedance sensing system (ECIS Zθ, Applied Biophysics). ECIS plates (96W20idf PET, Applied Biophysics) were pretreated with L-Cystein and coated with 1% gelatin. After taking baseline measurements, endothelial cells with and without knockdown or overexpression were added to the plate in the absence or presence of 10 μM Y-27632 ROCK inhibitor (StemCell technologies, 72302). Multiple frequency/time (MFT) mode was used for real-time assessment of the barrier. Results are expressed as relative resistance at frequency of 4,000 Hz corrected for baseline resistance. ECIS software was used for further mathematical modeling. Using impedance data, this model enables to calculate the cell morphological parameters cell-cell (Rb) and cell-matrix (α) contacts (27 (link), 28 (link)).
In addition, stable endothelial barriers were modulated by addition of 10 μM Y-27632 ROCK inhibitor or 1 μg/mL recombinant SEMA3A (R&D Systems, 1250-S3). Results are expressed as percentage of the average barrier of the endothelial cells measured over 4–5 h before addition of stimuli.

Vreeken D., Bruikman C.S., Stam W., Cox S.M., Nagy Z., Zhang H., Postma R.J., van Zonneveld A.J., Hovingh G.K, & van Gils J.M. (2021). Downregulation of Endothelial Plexin A4 Under Inflammatory Conditions Impairs Vascular Integrity. Frontiers in Cardiovascular Medicine, 8, 633609.

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Lentiviral-Mediated Generation of Human Induced Pluripotent Stem Cells

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A single lentiviral “stem cell cassette” has been used, flanked by loxP-sites (hSTEMCCA-loxP), and encoding for all four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) in a single polycistonic vector (Spitalieri et al., 2015 (link), 2018 (link)). Approximately 1.5 × 10⁵ HDFs have been seeded on plastic in 35 mm culture plates and infected in DMEM, containing 15% FBS. The medium has been replaced after 16 h with hiPSCs medium: Dulbecco’s Modified Eagle’s medium-F12 (DMEM/HAM’s F12) (Gibco, Waltham, MA, United States) with 20% knock out serum replacement (KSR) (Gibco, Waltham, MA, United States), 1 mM L-glutamine, 1% penicillin/streptomycin, 1% non-essential amino acid solution, 0.1 mM β-mercaptoethanol and 10 ng/ml of basic Fibroblast Growth Factor (bFGF) (PeproTech, London, United Kingdom) and changed every 2–3 days. hiPSCs colonies have been picked 20–25 days post-infection on the basis of morphology and expanded by plating on mitomycin C-treated MEFs in hiPSCs medium (Sigma-Aldrich, St. Louis, MO, United States). Successively, hiPSCs have been manually picked, passaged on human embryonic stem cell-qualified Matrigel-coated plates (0.05 mg/mL) (Corning, NY, United States) and cultured under feeder-free condition in mTeSR1 medium with Y-27632 ROCK inhibitor (StemCell, Canada), mantaining the stability over 20 and more passages.

Spitalieri P., Talarico R.V., Murdocca M., Fontana L., Marcaurelio M., Campione E., Massa R., Meola G., Serafino A., Novelli G., Sangiuolo F, & Botta A. (2018). Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2. Frontiers in Physiology, 9, 967.

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Embryoid Body Formation from hESCs

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Embryoid body (EB) formation from hESCs (H1 line) were performed using Aggrewell^TM800 plates (#27865, StemCell Technologies Inc.) and APEL2 medium (#05270, StemCell Technologies Inc.) following the guidelines of the manufacturer’s technical manual (#29146). In brief, single-cell suspensions were prepared by treatment of hES cells with accutase (#07920, StemCell Technologies Inc.), and 1 × 10⁶ cells were seeded per well of an Aggrewell^TM800 plate in APEL2 medium supplemented with 10 μM Y-27632 ROCK inhibitor (#72307, StemCell Technologies Inc.) followed by 72 h incubation with daily media change. Embryoid bodies were then embedded into Matrigel and differentiated in APEL2 medium for 6d. The medium was replaced every day.

Asmar A.J., Abrams S.R., Hsin J., Collins J.C., Yazejian R.M., Wu Y., Cho J., Doyle A.D., Cinthala S., Simon M., van Jaarsveld R.H., Beck D.B., Kerosuo L, & Werner A. (2023). A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development. Nature Communications, 14, 4499.

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Reprogramming Fibroblasts to hiPSCs

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Human dermal fibroblasts were reprogrammed into human-induced pluripotent stem cells (hiPSCs), as previously reported [34 (link),35 (link),36 (link),37 (link)]. A healthy donor subject (wild type, WT, 46 XX, age 46) has been recruited for dermal biopsy. Before participation, informed written consent was obtained. The project was approved by The Committees on Health Research Ethics of Tor Vergata Hospital (2932/2017) and in accordance with the Declaration of Helsinki.
hiPSC lines were manually picked, passaged on human embryonic stem cell-qualified Matrigel-coated plates (0.05 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA), and cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) with Y-27632 ROCK inhibitor (Stem Cell Technologies, Vancouver, Canada), and under a feeder-free condition, preserving the stability for over 30 and more passages. The stemness characteristics and karyotype were also verified (data not shown).

Spitalieri P., Centofanti F., Murdocca M., Scioli M.G., Latini A., Di Cesare S., Citro G., Rossi A., Orlandi A., Miersch S., Sidhu S.S., Pandolfi P.P., Botta A., Sangiuolo F, & Novelli G. (2022). Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids. Cells, 11(7), 1235.

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Differentiation of hiPSCs into Embryoid Bodies

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For human induced pluripotent stem cell experiments (hiPSC), REBL-PAT (non-disease) cells were used as established and characterised previously [49 (link)]. hiPSC were maintained in Essential 8 medium (E8) on tissue culture flasks coated with recombinant vitronectin peptide (VTN-N) following manufacturer's instructions. For hiPSC passage, cells incubated with TrypLE Express were collected in E8 supplemented with 10 μM Y-27632 ROCK inhibitor (72304, Stem Cell Technologies, UK). The hanging drop method used to generate embryoid bodies (EBs) from hiPSCs was adapted from [50 (link)]. hiPSCs were harvested 48 h after seeding and resuspended in E8 with 10 μM Y-27632 and 4 mg/mL polyvinyl alcohol (Sigma, UK). 20 μL droplets containing 2000 cells/droplet were pipetted onto the lid of a 10 cm petri dish containing 10 mL PBS to maintain hydration. The EBs were formed for 24 h at 37 °C, then collected in DMEM. EBs were allowed to sediment at the bottom of a 15 ml falcon tube for 10–15 min at 37 °C and were subsequently cultured in peptide gels maintained in Essential 6 medium (E6) to allow spontaneous differentiation.
All cell lines were maintained in antibiotic-free conditions, at 37 °C and 5% CO₂ in a humidified atmosphere. All media components were obtained from Gibco, UK unless specified.

Ashworth J.C., Thompson J.L., James J.R., Slater C.E., Pijuan-Galitó S., Lis-Slimak K., Holley R.J., Meade K.A., Thompson A., Arkill K.P., Tassieri M., Wright A.J., Farnie G, & Merry C.L. (2019). Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro. Matrix biology : journal of the International Society for Matrix Biology, 85-86, 15-33.

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Kidney Organoid Differentiation from iPSCs

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iPSCs were differentiated into kidney organoids following the previously published protocol by Freedman et al. (5 (link)) (Figure 1B). Briefly, iPSCs were dissociated with 1:3 Accutase and plated onto 24-well plates precoated with 0.5% GelTrex in mTeSR1 supplemented with 10 μM Y-27632 ROCK Inhibitor (STEMCELL Technologies). After 24 hours, another layer of GelTrex at 1.5% was added in mTeSR1 media. At the end of day 4, the medium was replaced with Advanced RPMI (Gibco) supplemented with 12 μM CHIR-99021 and 10 ng/ml noggin (STEMCELL Technologies). Approximately 60 hours later, the medium was changed to Advanced RPMI with B27 (Gibco). Organoids were cultured in this medium until collection at day 25.

Liu E., Radmanesh B., Chung B.H., Donnan M.D., Yi D., Dadi A., Smith K.D., Himmelfarb J., Li M., Freedman B.S, & Lin J. (2020). Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics. Kidney360, 1(3), 203-215.

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Single-Cell Sorting for Edited Cells

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Cells were dissociated using Accutase at room temperature, counted and transferred to a conical tube. Cells were collected by centrifugation at 200 g and washed with sterile sort buffer (Ca & Mg free PBS, 25 mM HEPES pH 7.0, 1–2.5 mM EDTA and 0.5% BSA or 1%–2% FCS). Cells were collected again and resuspended at a concentration of 5–8×10⁶ cells/ml. Untransfected cells were used for gating cell size on the FACS Aria cell sorter (BD) and edited cells then sorted into 150 μL DMEM/F12 + 20% FCS + 10% antibiotic and antimycotic + 10 μM Y-27632 ROCK inhibitor (STEMCELL Technologies) into each well of a 96 well plate.

Nazlamova L., Villa Vasquez S.S., Lord J., Karthik V., Cheung M.K., Lakowski J, & Wheway G. (2022). Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations. Frontiers in Genetics, 13, 1009430.

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Generating Marfan Syndrome iPSCs

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Healthy donor subject (wild type, WT) and Marfan Syndrome (MFS) patient underwent skin biopsy, following written informed consent.
The project was approved by The Committees on Health Research Ethics of Tor Vergata Hospital (2932/2017) and in accordance with the Declaration of Helsinki. Diagnosis of MFS is based on both Ghent clinical criteria and genetic test revealing a rare FBN1 gene deletion (haploinsufficiency).
Human fibroblasts reprogramming was performed, as previously reported.37 (link),38 (link) hiPSC colonies were picked 20–25 days post infection on the basis of morphology and expanded by plating on mitomycin C-treated MEFs in hiPSCs medium. Successively, hiPS lines MFS and WT were manually picked, passaged on human embryonic stem cell-qualified Matrigel-coated plates (0.05 mg/mL; BD Biosciences) and cultured under feeder-free condition in mTeSR1 medium (Stem Cell Technologies) with Y-27632 ROCK inhibitor (Stemcell Technologies), maintaining the stability over 20 and more passages. The stemness propriety and karyotype analysis were also verified (data not shown).

Spitalieri P., Marini M., Scioli M.G., Murdocca M., Longo G., Orlandi A., Novelli G, & Sangiuolo F. (2021). Effects of Simulated Microgravity on Wild Type and Marfan hiPSCs-Derived Embryoid Bodies. Cellular and Molecular Bioengineering, 14(6), 613-626.

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