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R1

SCRC-1011

Product category
Animal cells
Organism
Mus musculus, mouse
Classification
Eukaryota, Animalia, Metazoa, Chordata, Vertebrata, Tetrapod
Cell type
embryonic stem cell
Morphology
Spherical colony
Tissue
Embryo; Inner cell mass
Applications
Stem cell research
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

ATCC determines the biosafety level of a 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 understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

Characteristics

Growth properties
Adherent
Passage history
Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). They were also tested by diploid embryo <->
Derivation

The R1 cell line was established in August 1991, from a 3.5 day blastocyst produced by crossing two 129 substrains (129S1/SvImJ and 129X1/SvJ).


Gender
Male
Strain
129X1 x 129S1
Karyotype
The cells are heterozygous for the c locus (+/c (ch)) and for the pink eye locus (+/p). In the F1 generation the coat color is uniform agouti, while in the F2 these two coat color genes segregate. The segregation could result in several coat types, from albino, through light brown, to black, depending on the genetic background of the partner of the germline chimaera.
Comments

Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras (Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547). At early passages (up to passage #14), one third of the completely R1-derived newborns generated by tetraploid embryo <-> R1 aggregates survived. No live offspring were produced from cells older than passage #14.*

However, about 20% of subclones derived from passage #14 had the original developmental potential of R1 when tested by tetraploid aggregates (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). R1-derived animals reached adulthood and were fertile. The genetically altered lines derived from R1 gave high efficiency of germline transmission either by injecting them into C57 blastocyst or aggregating them with CD-1 or ICR outbred 8-cell stage embryos. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras.

*Current ATCC stocks of R1 cells are beyond passage 14. Current stocks of alternative subclone of R1 cells, designated R1/E (ATCC SCRC-1036), are below passage 14 and have been shown to be germline competent.


Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107)
3. 10% to 15% ES-Cell Qualified FBS (ATCC® SCRR-30-2020) or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Feeder cells
Feeder cells may be grown in medium containing fewer growth factors than those required by the ES cells. Feeder cells are available from ATCC. Consult the product detail page and the product sheet provided for the feeder cells you wish to use for medium requirements. Feeder cells should be initiated 24 to 48 hours prior to inoculating with embryonic stem (ES) cells.
Handling procedure

Complete Medium for Feeder Cells

Feeder cells may be grown in medium containing fewer growth factors than those required by the ES cells. Feeder cells are available from ATCC. Consult the product sheet provided for the feeder cells you wish to use for medium requirements. 

Feeder cells should be initiated 24-48 hours prior to inoculating with embryonic stem (ES) cells.

Feeder Cells

ATCC recommends culturing R1 on mouse embryonic fibroblasts (MEFs) that have been mitotically arrested by either irradiation or treatment with Mitomycin-C. R1 cells have been cultured on mitotically arrested MEF (CF-1) (ATCC® SCRC-1040™).

  1. At least one day before plating the ES cells, prepare the desired combination of flasks with feeder cells to accommodate an initial ES cell seeding density of 30,000 cells/cm2 to 50,000 cells/cm2.
  2. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from this lot of ATCC® SCRC-1011™.
  3. Plate mitotically arrested mouse embryonic fibroblasts (MEFs) as a feeder layer at approximately 55,000 feeder cells/cm2 in complete medium for feeder cells. 
  4. Refer to the product sheet for mitotically arrested MEF for detailed handling instructions.

Feeder cells should be used within one week of plating. It is best to use feeder cells within 24-48 hours of initiation.

Embryonic Stem (ES) Cells

  1. 30 Minutes Prior to Handling Cells – Pre-warm complete growth medium for ES cells at 37°C for at least 30 minutes before adding to cells.
  2. One Hour Prior to Thawing the ES Cells – Perform a 100% medium change for the MEFs using complete growth medium for ES cells.
  3. Thaw the vial of ES cells by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 90 seconds).
  4. Remove the vial from the water bath before the contents are completely thawed, and decontaminate by dipping in or spraying with 70% ethanol.
  5. All of the operations from this point on should be carried out under strict aseptic conditions.
  6. Transfer the vial’s contents plus 5 mL of complete growth medium for ES cells to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth medium for ES cells to bring the total volume to 10 mL.
  7. Spin the cells at 270 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 mL of complete growth medium for ES cells.
  8. Add the 2 mL of cell suspension to the appropriate size flask containing feeder cells and fresh compelte growth medium for ES cells (see batch specific information). ES cells should be plated at a density of 30,000 – 50,000 cells/cm2.
  9. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. 

Routine Handling

Perform a 100% medium change every day. Passage the cells every 1 to 2 days. If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation.

Make sure that you have prepared a sufficient number of flasks pre-plated with MEF feeder layers to support frequent passage of the ES cells.

Subculturing procedure
Subculturing Procedure

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

  1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
  2. One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Dissociation and Transfer of ES Cells

  1. Aspirate the medium from the flask(s) containing ES cells.
  2. Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
  3. Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
  4. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
  5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
  6. Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.
  7. Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
  8. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.
Reagents for cryopreservation
Complete growth medium supplemented with 10% (v/v) FBS and 10% (v/v) DMSO (ATCC 4-X)

Quality control specifications

Mycoplasma contamination
Not detected

History

Depositors
A Nagy
Year of origin
1991
Special collection
National Stem Cell Resource

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

Material Transfer Agreement Addendum for Mount Sinai R1

Nonprofit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Mount Sinai R1. We cannot ship this item until we receive this addendum.

Email the addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

For-profit organizations
For every order of this item, you must have a research-use license from the material contributor. We cannot ship this item until we receive this license or authorization directly from the contributor in the form of an email. We are providing the following contact information for obtaining this license, but this information may change without notice:

Mount Sinai Hospital
Attn: Michael Hanna, Sr. Business Development Officer
Office of Technology Transfer and Industry Liason
Mount Sinai Hospital, part of Sinai Health System
Room 843 - 600 University Avenue, Toronto, Ontario, Canada M5G 1X5
Phone: (416) 586-8225
Email: [email protected]

If sending the license to us, email the license to [email protected] with a reference to both your account and sales order numbers. Once either the license or authorization is received, your order will be reviewed, and this item will be released for shipment if all requirements are met. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

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

Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314

Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547

Nagy A, Rossant JProduction and analysis of ES-cell aggregation chimerasIn: Nagy A, Rossant JGene Targeting: A Practical ApproachOxfordOxford University Press177-206, 1999

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

View All Curated Citations for this Product

Frequently Asked Questions

Need assistance with this product? Contact our Technical Support team.

Telephone

Telephone

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800-638-6597

Outside the US

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