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ROCK Inhibitor Y27632

ACS-3030


Volume: 10 mg
Storage: -20°C

ROCK Inhibitor Y-27632 is a cell permeable potent and selective inhibitor of the Rho-associated protein kinase (ROCK). Treatment with ROCK Inhibitor Y-27632 increases the survival of many types of stem cells in vitro.

This product has been qualified for use with 3D culture of organoids, hESCS/hiPSCs, and conditionally reprogrammed cells (CRC).


Structure of ROCK Inhibitor Y27632


Chemical name: trans-4-[(1R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride


Product category
Reagents
Comments
Purity is > 98% as confirmed by HPLC analysis. Structure and mass are confirmed by NMR and mass spectrometry.
Reconstituted product can be aliquoted and stored at -20°C for up to six months. Protect from light.
Applications
Stem cell research
Cell growth and viability
Storage conditions
-20°C or colder
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Documentation

ATCC determined that a biosafety level is not applicable to this material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to complete your own risk assessment and understand any potential hazards associated with the material per your organization’s policies and procedures and any other applicable regulations as enforced by your local or national agencies.

Detailed product information

General

Specific applications
  • Increased primary human organoid formation efficiency and recovery from thawing and passaging.
  • Enhanced survival and maintenance of pluripotency of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC)1.
  • Critical media component in conditional reprogramming of cells (CRC) culture technology2.

Handling information

Handling procedure

Preparation and storage

Prepare a 10 mM stock solution

  1. Aseptically add 3 mL of sterile water (ATCC 60-2450) or D-PBS (ATCC 30-2200) to the 10 mg vial of ROCK Inhibitor Y27632. Mix thoroughly by pipetting.
  2. Aliquot the stock solution in working volumes based on routine use.
  3. Store aliquots at -20°C to -80°C and avoid repeated freezing and thawing. Once thawed, aliquots may be kept at 2°C to 8°C for two weeks.
A. Organoid culture
  • Use at a final concentration of 10 µM (1 µL of a 10 mM stock solution per mL of complete culture media).
  • Supplement the media for the first 2-3 days post-thaw and during the first 2-3 days post-passaging.   
  • Add ROCK Inhibitor Y-27632 directly to complete culture media as needed. Do not store culture media once supplemented.

B. Conditional reprogramming of cells (CRC) culture

  • Utilized as a component in F-media. Use with or without feeder cells.
  • Use at a final concentration of 10 µM (1 µL of a 10 mM stock solution per mL of complete culture media)
C. Human induced pluripotent stem cells (iPSC) culture

Refer to the ATCC Stem Cell Culture Guide for more information.

Cell culture medium: Pluripotent Stem Cell SFM XF/FF (ATCC ACS-3002) is recommended for feeder free culture and ROCK Inhibitor Y-27632 is required for initial culture of iPSCs.

Preparation of Medium Supplemented with ROCK Inhibitor Y-27632

  1. Thaw 10 mM stock solution of ROCK Inhibitor Y-27632 on ice.
  2. Dilute 10 mM ROCK Inhibitor Y-27632 1:1000 in cell culture medium to obtain a final concentration of 10 μM. For example, if you are preparing 500 mL of media add 0.5 ml 10 mM ROCK inhibitor
  3. Medium supplemented with ROCK Inhibitor Y-27632 must be used immediately.

Handling Procedure for Frozen Cells and Initiation of Cultures

  1. 30 Minutes Prior to Handling Cells ­ Pre­warm the appropriate stem cell culture medium at 37°C for at least 30 minutes before adding to cells.
  2. Remove cryovial of frozen cells from liquid nitrogen storage.
  3. Thaw the cells by gentle swirling in a 37°C water bath. To reduce the possibility of contamination, keep the O­ring and cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes). Remove the cryovial from the water bath when only a few ice crystals are remaining.
  4. Sterilize the cryovial by rinsing with 70% ethanol.
  5. Using a 1 mL or 5 mL pipette, gently transfer the cell suspension to a 15 mL conical tube.
  6. Slowly add 4 mL stem cell culture medium including 10 µM of ROCK Inhibitor Y-27632 (ATCC ACS­-3030) dropwise, to the conical tube. Use an additional 1 mL of media to rinse the cryovial and transfer the liquid to the 15 mL conical tube. Shake the conical tube gently to mix the cells while adding media.
  7. Gently pipette the cells up and down 1-2 times to mix thoroughly. Avoid breaking apart the aggregates into a single­cell suspension.
  8. Centrifuge the cells at 200 x g for 5 minutes.
  9. Aspirate the supernatant and discard. Gently tap on the bottom of the tube to loosen the cell pellet.
  10. Add 1 mL of stem cell culture medium that has been supplemented with ROCK Inhibitor Y27632 (ATCC ACS-­3030) to a final concentration of 10 µM. Gently resuspend the pellet by pipetting up and down 1-2 times with a 1 mL tip, maintaining the cell aggregates.
  11. Plate the cells as desired under  feeder­free culture condition
  12. ROCK Inhibitor Y-27632 is not necessary in subsequent cell culture medium changes and is not required for passaging cells.

Note: The use of ROCK inhibitor may cause a transient spindle-like morphology effect on iPSCs. However, the colony morphology will recover after subsequent media change without ROCK inhibitor


References

  1. Li X. et al, ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod 24(3): 580-589, 2009. PubMed: 19056770
  2. Liu, Xuefeng, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180(2): 599-607, 2012.

Quality control specifications

Mycoplasma contamination
Not detected

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Bergqvist C, et al. An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells. Stem Cell Res 23:33-38, 2017. PubMed: 28668644

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