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™) withxa0 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.

Volumes used in this protocol are for 75 cm²xa0flasks; 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⁺⁺xa0free Dulbeccos phosphate-buffered saline (D-PBS) (ATCCxa030-2200) or 0.25% (w/v) Trypsin - 0.53 mM EDTA (ATCCxa030-2101) 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).
Note:xa0To 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.
3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
5. Discard supernatant. Resuspend the cell pellet in fresh growth medium.
6. Add appropriate aliquots of the cell suspension to new culture vessels
7. Incubate cultures at 37°C.

xa0

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

Medium Renewal: Once between subcultures if necessary

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