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C3H/10T½-mRuby clone 2


Product category
Animal cells
Mus musculus, mouse
Eukaryota, Animalia, Metazoa, Chordata, Vertebrata, Tetrapod
3D cell culture
Product format
Storage conditions
Vapor phase of liquid nitrogen
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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.

Cells contain cytomegalovirus (CMV) DNA sequences

Cells contain SV40 sequences

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


Specific applications
Fluorescently-labeled, mesenchymal stem cell-like cells for musculoskeletal research. Cells may also be used in co-cultures with human umbilical vein derived endotheilial cells (HUVECS) for angiogenesis research.


1.0 mL
Growth properties

Cells were transfected with pVitro2-mRuby2-blast plasmid using JetPrime (polyplus) transfection reagent. Stably-transfected cells were selected for by using 3 µg/mL blasticidin for 10 days. Clones were selected using flow cytometry and expanded. A specific clone was selected for subsequent testing and deposit.


CRL-3268 were generated by transfecting C3H10T1/2; cells (ATCC® CCL-226™) with  fluorescently labeled mRuby2 protein. They are similar to untransfected C3H10T1/2; cells in terms of cell proliferation, osteogenic, chrondrogenic and adipogenic differentiation. They remain sensitive to post confluence inhibition of cell division.

These cells were stablely transfected with the mRuby2 gene via plasmid transfection and 3 µg/mL blasticidin selection for 10 days. This clone was isolated using flow cytometry.

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
The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
95% Air, 5% CO2
Handling procedure
1 amp --> 1 T-75

Thaw ampoule in 37˚C water bath for approximately 2 minutes.  Transfer thawed cell suspension to a 15.0 mL centrifuge tube containing 9 mL complete medium.  Mix suspension by gentle inversion.  Remove 0.5-1.0 mL for cell count. Centrifuge remaining suspension in the 15mL centrifuge tube at 175-195 x g (1000rpm in an IEC HN SII centrifuge or equivalent) for 5 minutes, RT˚. Discard supernatant and gently resuspended pellet in 5ml fresh complete medium. Transfer 5 ml re-suspended cells into 1 T-75 flask containing 10ml fresh medium. Place the cells in a 5% CO2 incubator @ 37˚C

Subculturing procedure

Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% (w/v) Trypsin - 0.53 mM EDTA (ATCC 30-2101) solution to remove all traces of serum which contains trypsin inhibitor.
  2. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
  3. Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
  6. Discard supernatant. Resuspend the cell pellet in fresh growth medium.
  7. Add appropriate aliquots of the cell suspension to new culture vessels
  8. Incubate cultures at 37°C.


Subcultivation Ratio: Seed new flasks at 2000 viable cells/cm2.

Medium Renewal: Once between subcultures if necessary

Culture maintenance
Cultures are grown @ 37°C in a 95% air, 5% CO2 environment. Medium change every 2-4 days.
Reagents for cryopreservation
Complete growth medium supplemented with 10% (v/v) DMSO (ATCC 4-X)


E Ker, Stanford University
Year of origin

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.

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.


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

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.



Curated Citations

Reznikoff CA, et al. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res. 33: 3239-3249, 1973. PubMed: 4796800

Reznikoff CA, et al. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33: 3231-3238, 1973. PubMed: 4357355

Lam AJ, et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9(10): 1005-1012, 2012. PubMed: 22961245

Ker DF, et al. Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering. PLoS One 10(9): e0139054, 2015 PubMed: 26407291

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