Y27632

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

Y27632 is a chemical compound that functions as a Rho-associated protein kinase (ROCK) inhibitor. It is commonly used in cell culture and research applications to modulate cellular processes, such as cell contractility and migration.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

ROCK inhibitor Y27632 (5 citations)

79 protocols using y27632

Murine Prostate Organoid Culture and Maintenance

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Murine prostate organoids were established and maintained as previously described^{18 (link),19 (link)}. Full prostate organoid media composed of: 1X ADMEM, 10 mM HEPES, 1X Glutamax, 0.5X Pen/Strep, 1X B27 (Fisher Scientific, #17504–044), 1.25 mM N-acetylcysteine (Sigma, A9165–100G), 10 mM Nicotinamide (Sigma, N0636–500G), 500 nM A83–01 (Tocris, #2939), 5 ng/mL recombinant murine EGF (Peprotech, #315–09), 10 ng/mL recombinant NRG1 (Peprotech, #100–03), 1 nM Dihydrotestosterone (Selleck Chemicals, S4757) 10% NOGGIN conditioned media, 5% RSPO-I conditioned media. Single cell embedded prostate organoids were supplemented with 10 μM Y-27632 (Fisher Scientific, #50–863-7) for the first 2–3 days in culture prior to change with fresh organoid media lacking Y-27632. Ex-vivo transformed organoids were seeded at 3E3 cells per 20 μL matrigel dome and passaged every 3–4 days. Wild-type organoids were seeded at 10E3 cells per 20 μL matrigel dome and passaged every 5–7 days. For monolayer adaptation, western blot validated knockout organoids were dissociated using methods described above and seeded at 1E5 cells/mL in full organoid media containing 10 μM Y-27632 on a collagen I coated 10 cm plate (Fisher Scientific, #08–772-75). After 5 days adaptation and expansion, cells were processed for protein lysates, or dissociated for orthotopic transplantation.

Romero R., Chu T., González-Robles T.J., Smith P., Xie Y., Kaur H., Yoder S., Zhao H., Mao C., Kang W., Pulina M.V., Lawrence K.E., Gopalan A., Zaidi S., Yoo K., Choi J., Fan N., Gerstner O., Karthaus W.R., DeStanchina E., Ruggles K.V., Westcott P.M., Chaligné R., Pe’er D, & Sawyers C.L. (2024). The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1. bioRxiv.

+ Open protocol

+ Expand

CRISPR-Mediated Gene Knockdown in Human Bronchial Epithelial Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Each gRNA sequence (Table S3) was selected using CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) and GuideScan⁹¹ (link) and was used to design a pair of complementary 5′-phosphorylated oligonucleotides (5′-PO₄-ATGNNNNNNNNNNNNNNNNNNNNGTTTCAGAGC-3′ and 5′-PO₄-TTAGCTCTGAAACNNNNNNNNNNNNNNNNNNNNCATGTTT-3′; where Ns indicate sequences that vary according to the gRNA). A G was added to the 5′ ends of gRNA sequences not beginning with a G. gRNA oligonucleotides (oligos; IDT, San Jose, CA) were annealed and cloned into the hU6-sgRNA-cr3-EF1a-Puro plasmid digested with BstXI and BlpI (New England Biolabs). HBECs were initially transduced with EF1a-dCas9-KRAB-P2A-BSD lentivirus and maintained in BEGM with 10 μM Y-27632 and 10 μg/mL blasticidin (Thermo Fisher Scientific) for 3 d for selection. Cells were subsequently transduced with sgRNA lentivirus and maintained in BEGM with 10 μM Y-27632, 10 μg/mL blasticidin, and 1 μg/mL puromycin (Thermo Fisher Scientific) for 3 d for selection. Transduced and selected cells were then passaged to Transwell inserts for culture at ALI for 23 d. Where indicated, IL-13 (10 ng/mL) was added for the final 7 d of culture.

Koh K.D., Bonser L.R., Eckalbar W.L., Yizhar-Barnea O., Shen J., Zeng X., Hargett K.L., Sun D.I., Zlock L.T., Finkbeiner W.E., Ahituv N, & Erle D.J. (2022). Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma. Cell Genomics, 3(1), 100229.

+ Open protocol

+ Expand

Glioblastoma Cell Line Culture

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Three glioblastoma cell lines were used: U87-MG (ATCC, Manassas, VA), primary glioblastoma cell line SMC448 (kindly provided by Dr. Do-Hyun Nam, Samsung Medical Center, Seoul, South Korea), and JX12. JX12 is a classical subtype patient-derived GBM xenograft cell line (xenoline) that was established as previously described [21 (link)] in immunocompromised athymic nude mice from surgical resection waste specimens obtained from consented patient undergoing surgical therapy for primary GBM at the University of Alabama at Birmingham Comprehensive Cancer Center Brain Tumor Tissue Core Facility under the approval of annually renewed IRB (approval no. X050415007). The cells were grown in three-dimensional tumorsphere culture in Neurobasal media supplemented with 1 mM glutamine (Life Technologies, Carlsbad, CA), 8 μg/mL heparin (JT Baker, Phillipsburg, NJ), 0.5X N2 (Gibco, Grand Island, NY), 0.5X B27 (Gibco), 1% Penicillin/Streptomycin (Corning, Manassas, VA), 20 ng/mL EGF (Shenandoah Inc., Warwick, PA), and 10 ng/mL FGF (Shenandoah Inc) (NBE media). For the Y-27632 and fasudil experimental groups, NBE was supplemented with either 45 μM Y-27632 (Thermo Fisher Scientific, Pittsburg, PA) or 10 μM fasudil hydrochloride (Biotang Inc., Lexington, MA), respectively.

Tilson S.G., Haley E.M., Triantafillu U.L., Dozier D.A., Langford C.P., Gillespie G.Y, & Kim Y. (2015). ROCK Inhibition Facilitates In Vitro Expansion of Glioblastoma Stem-Like Cells. PLoS ONE, 10(7), e0132823.

+ Open protocol

+ Expand

Genetic Manipulation of HBECs using KRAB-dCas9

Check if the same lab product or an alternative is used in the 5 most similar protocols

Enhancer sequences (Table S5) were PCR-amplified from genomic DNA from BEAS-2B cells or synthesized (IDT) and cloned into XbaI and SbfI sites of the mP-KRAB-dCas9_EF1a-BSD plasmid using the Quick Ligation Kit or NEBuilder HIFI Assembly. HBECs were initially transduced with enhancer-mP-KRAB-dCas9 lentivirus and maintained in BEGM with 10 μM Y-27632 and 10 μg/mL blasticidin (Thermo Fisher Scientific) for 3 d for selection. Cells were subsequently transduced with sgRNA lentivirus and maintained in BEGM with 10 μM Y-27632, 10 μg/mL blasticidin, and 1 μg/mL puromycin (Thermo Fisher Scientific) for 3 d for selection. Transduced and selected cells were then passaged to Transwell inserts for culture at ALI for 23 d. Where indicated, IL-13 (10 ng/mL) was added for the final 7 d of culture.

+ Open protocol

+ Expand

Intestinal Crypt Cell Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Crypts from each intestinal segment were independently incubated with Dispase (36 U; Corning, Corning), Y‐27632 (10 mM), and DNase I (300 μg; Alfa Aeser) in 1× Hank's Balanced Salt Solution (HBSS) for 10 min at 37°C. Single cells were washed with 1× HBSS, resuspended in flow cytometry buffer (2% Bovine Serum Albumin in 1× PBS), and passed sequentially through 100 μm, 70 μm, and 40 μm filters. Cells were then stained with Propidium Iodide (BioLegend) for FACS sorting.

Schaaf C.R., Polkoff K.M., Carter A., Stewart A.S., Sheahan B., Freund J., Ginzel J., Snyder J.C., Roper J., Piedrahita J.A, & Gonzalez L.M. (2023). A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease. The FASEB Journal, 37(6), e22975.

+ Open protocol

+ Expand

Intestinal Crypt Cell Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Crypts from each intestinal segment were independently incubated with Dispase (36 U; Corning, Corning), Y-27632 (10 mM), and DNase I (300 μg; Alfa Aeser) in 1× Hank’s Balanced Salt Solution (HBSS) for 10 min at 37°C. Single cells were washed with 1× HBSS, resuspended in flow cytometry buffer (2% Bovine Serum Albumin in 1× PBS), and passed sequentially through 100 μm, 70 μm, and 40 μm filters. Cells were then stained with Propidium Iodide (BioLegend) for FACS sorting.

Smith W.C., Price D.A., Greenberg R.M, & Battelle B.A. (1993). Opsins from the lateral eyes and ocelli of the horseshoe crab, Limulus polyphemus.. Proceedings of the National Academy of Sciences of the United States of America, 90(13), 6150-6154.

+ Open protocol

+ Expand

Organoid Culture of Mouse Pancreatic Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The mouse pancreatic tissues from KC/tdPd mice treated with the GW diet (50 mg/kg) or control diet for 3 days were digested with digestion buffer (1 mg/ml collagenase IV [Sigma-Aldrich] and 10 U/ml DNase I [Sigma-Aldrich] in DMEM) at 37 °C for 30 min, and the pancreatic epithelial cells were harvested, counted, and embedded in Matrigel (Corning). The Matrigel was topped with organoid culture medium comprising advanced DMEM/F-12 supplemented with penicillin–streptomycin, 2 mM GlutaMAX, 10 mM HEPES (all from Thermo Fisher Scientific), mouse recombinant Wnt-3A at 100 ng/ml (MilliporeSigma), mouse epidermal growth factor at 50 ng/ml (Invitrogen), mouse Noggin at 100 ng/ml (PeproTech), human R-spondin-1 at 1 µg/ml (PeproTech), N-acetyl-L-cysteine at 1 mM (Sigma-Aldrich), 1× N-2 supplement (Thermo Fisher Scientific), 1× B-27 supplement (Thermo Fisher Scientific), and Y-27632 at 10 µM (Fisher Scientific). PPARδ agonist GW (1 μM; Sigma-Aldrich) was added to organoid cultures for the mice treated with the GW diet. Organoids were imaged on days 1, 3, and 6 of culture unless otherwise specified. The conditioned culture media were harvested and passed through a 0.45-µm filter to remove the cell debris before being used for further studies.

Liu Y., Deguchi Y., Wei D., Liu F., Moussalli M.J., Deguchi E., Li D., Wang H., Valentin L.A., Colby J.K., Wang J., Zheng X., Ying H., Gagea M., Ji B., Shi J., Yao J.C., Zuo X, & Shureiqi I. (2022). Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ. Nature Communications, 13, 2665.

+ Open protocol

+ Expand

Cell Migration Dynamics on Guidance Cues

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were plated at 40,000-50,000 cells ml^-1 in 2 ml of media in 35 mm dishes. MDA-MB-231 cells were incubated for 2 hrs supplemented with blebbistatin (Sigma Aldrich), ML-7 (Sigma Aldrich), Y-27632 (Calbiochem, Billerica, MA, USA), P5D2 β1 integrin blocking antibody (mouse mAb, ascites, from Mark Ginsberg, University of California, San Diego) [41 (link)], calyculin A (Santa Cruz Biotechnology, Dallas, TX, USA) and MnCl₂ (Fisher) and MTLn3 cells were incubated for 12 hrs with supplemented with calyculin A, MnCl_2, Y-27632, and P5D2 on contact guidance cues in imaging media. Substrates with attached cells were inverted onto two strips of double sided tape attached to a microscope slide to generate a flow chamber. The chamber was filled with imaging media and sealed with a 1:1:1 mixture of vasoline, lanolin and paraffin wax. Chambers were imaged by phase contrast microscopy on a heated stage at 37°C every 2 min for 12 hrs. Images were captured at 10× (NA 0.50, Nikon) as described above.
Cell centroids were identified and tracked manually using the MTrackJ plugins of ImageJ (National Institutes of Health, Bethesda, MD, USA). Cell speed and directionality were calculated over a time lag of 2 min averaged over 12 hrs as described in a previous paper [15 ].

Wang J, & Schneider I.C. (2016). Myosin Phosphorylation on Stress Fibers Predicts Contact Guidance Behavior across Diverse Breast Cancer Cells. Biomaterials, 120, 81-93.

+ Open protocol

+ Expand

Astrocyte Scratch Injury Assay and Recovery

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

A scratch injury ~600µm in diameter across a confluent astrocyte monolayer was made using a sterile P200 pipette tip[11 , 19 (link)]. At varying times after injury, cells were fixed and ICC performed. Treatments included: IL-1β (10 ng/ml; Peprotech)[8 (link), 11 ]; rm-TIMP-1 (10ng/mL; R&D) or the TIMP-1 C-terminal domain peptide (amino acids 126–184; Anaspec Inc.)[2 (link)]; GM6001 (12.5 µmol/L; Calbiochem)[20 (link)]; ROCK-inhibitor, Y-27632 (10 µM; Fisher)[8 (link)]. Scratch injuries were measured perpendicular to the longitudinal axis of the scratch at a minimum of three points spanning the width of the scratch. Measurements were then used to determine the amount of recovery relative to baseline (i.e. wound diameter at time of the scratch, or t=0) for each sample and treatment. The average of each technical replicate was then compared across biological replicates to assess variability, though all data points were included in the final analyses.

Johnson K.M, & Crocker S.J. (2015). TIMP-1 Couples RhoK Activation to IL-1β-induced Astrocyte Responses. Neuroscience letters, 609, 165-170.

+ Open protocol

+ Expand

Isolation of Intestinal Crypts

Check if the same lab product or an alternative is used in the 5 most similar protocols

Jejunum and colon tissues (∼2–3 cm in length) were opened longitudinally and washed with cold PBS. Tissues were transferred to a Petri dish filled with cold PBS and gently scraped with a microscope slide to remove mucus and jejunum villi before transfer to a dissociation solution [PBS containing 9 mM EDTA (Fisher Scientific, cat# AM9260G), 3 mM 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, cat# 10197777001), 10 μM ROCK inhibitor Y27632 (ATCC, cat#ACS-3030] and 1% penicillin/streptomycin). After 30 min of incubation at room temperature on a rotating shaker, tissues were transferred in a Petri dish filled with cold PBS. Crypts were isolated from the tissue by firm scraping with a microscope slide and filtered with a 100 μm cell strainer. After centrifugation (500 g, 5 min, 4°C) the pellet was re-suspended in cold Dulbecco’s Modified Eagle Medium (DMEM, Fisher Scientific, cat#31966047) supplemented with 10 µM Y27632 and crypts were counted with an hemocytometer. An aliquot was centrifuged (500 g, 5 min, 4°C) and the crypt pellet was lyzed in TRI Reagent (Zymo Research, cat# R2050) and stored at −80°C until RNA purification.

Mussard E., Lencina C., Gallo L., Barilly C., Poli M., Feve K., Albin M., Cauquil L., Knudsen C., Achard C., Devailly G., Soler L., Combes S, & Beaumont M. (2022). The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids. Frontiers in Cell and Developmental Biology, 10, 983031.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!