传代细胞方法说明书

Supplies:

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 37^oC.

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.