transfection host (Roche FuGENE® Transfection Reagents)

BD1X

H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.
Myoblastic cells in this line will fuse to form multinucleated myotubes and respond to acetylcholine stimulation.
Fusion occurs faster if the serum concentration in the medium is reduced to one percent.

Protocol: The myoblastic population will become depleted rapidly if the cultures are allowed to become confluent.
To prevent loss of myoblastic cells, cultures should be subcultured before they become confluent, and the line should be recloned periodically with selection for myoblastic cells.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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: 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. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium renewal: Every 2 to 3 days

Recommended medium (without the additional supplements or serum described under ATCC Medium): ATCC 30-2002
recommended serum: ATCC 30-2020