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These cell lines are useful in studying mitochondrial biology and the role of mitochondrial fusion in cell physiology.

MEFs were derived from embryos 10.5 days gestation. Embryos were mechanically dispersed by repeated passage through a P1000 pipette tip and plated with MEF medium (DME, 10% FCS, 1X nonessential amino acids, 1 mM L-glutamine (Invitrogen/Gibco)). Cells with deletions of Mfn1 (ATCC CRL-2992) or Mfn2 (ATCC CRL-2993) or both Mfn1/Mfn2-null MEFs (ATCC CRL-2994) were subsequently immortalized by transduction with SV-40 T antigen.

Donor Organism Characteristics: knockout

Both Mfn1 and Mfn2 are essential for mitochondrial fusion which is essential for embryonic development. 

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.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's 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 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.

Medium renewal: Every 2 to 3 days

Freeze medium: complete growth medium, 90%; DMSO, 10%

liquid nitrogen vapor phase

Temperature: 37°C

Atmosphere: air, 95%; carbon dioxide (CO₂), 5%