Cells are immortalized MEFs (genotype:xa0bad^-/-), which werexa0generated from BAD knockout mice.xa0

BAD is a pro-apoptotic BCL-2 family member (“BH3-only” subset) and is regulated by phosphorylation in response to growth/survival factors

This cell line is useful to study molecular mechanism of cell apoptosis, BCL-2 family pathway, and BAD function.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 1.0 to 2.0 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. An inoculum of 7 X 10³ to 1.5 X 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C. At subculture, cell concentration is between 8 X 10⁴ to 1.5 X 10⁵ cells/cm².

Danial N, et al. BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424 (6951) : 952-6, 2003 PubMed: 12931191

Cheng EH, et al. BCL-2, BCL-XL Sequester BH3 Domain-Only Molecules Preventing BAX- and BAK-Mediated Mitochondrial Apoptosis. Molecular Cell 8(3) : 705–711, 2001 PubMed: 11583631