上海誉贸实业有限公司
2014/3/31 9:55:51Supplies:
Modified Eagle’s Minimum Essential Medium
· Earle’s Balanced Salt Solution (BSS)
· 2 mM L-glutamine
· 0.1 mM nonessential amino acids
· 1.5 g/L sodium bicarbonate
· 1.0 mM sodium pyruvate
· 20% Fetal Bovine Serum (FBS)
· 1% Penicillin-Streptomycin (pen/strep) antibiotic solution
0.25% (w/v) Trypsin – 0.03% (w/v) EDTA
(1) – 10 mL aliquot kept in freezer
Phosphate Buffer Solution (PBS) – optional
Kept in refrigerator in cell culture lab (~450 mL)
15 mL sterile centrifuge tube
75 cm2 sterile tissue culture flasks
Method:
1. Heat trypsin and media to 37 degrees Celsius in water bath.
2. In laminar flow hood, aspirate media with glass Pasteur pipet connected to house vacuum line.
3. Add 5 mL phosphate buffer solution (PBS) or 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA to remove traces of FBS and residual Ca2+ that will deactivate EDTA. Immediay aspirate off solution.
4. Add 3 mL 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA
5. Incubate for ~5 minutes at 37oC.
6. When cells have detached, add 7 mL media to quench trypsin. Reduce clumping by forcefully pipetting mixture against side of flask 4-5 times.
7. Pipette total volume into 15 mL centrifuge tube. Centrifuge for 10 minutes at 1.3 x 1000 rpm.
8. Remove supernatant.
9. Vigorously resuspend Caco-2 cell pellet with 4/6/10 mL media (for 1:4, 1:6, 1:10, respectively).
10. Add 14 mL to each new 75 cm2 flask.
