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Cell Biology > Protocols
Enzymatic Dissociation of Rat Mesenteric Artery
1. Male C-D Strain Sprague-Dawley rats sacrificed by cervical dislocation.
2. Superior Mesenteric Artery excised, en bloc, from its origin at the aorta to the border of the intestine. Placed in ice-cold Hanks balanced salt solution in petri dish.
3. Fat, adventitia and veins removed by blunt dissection with care being exercised to minimize trauma to artery.
4. Cleaned mesenteric artery arcades (usually 4 per preparation) transferred into Enzyme Digest Solution (pH 7.4, filter-sterilized, prewarmed to 37°C) consisting of:
- 12 ml Eagles Minimum Essential Medium (Biowhittaker, Walkersville, MD #12-126B)
(spinner formulation with 0.2 mM CA2+ added).
- 24 mg Crystallized Bovine Serum Albumin (Pentex, Miles Laboratories, Elkhart,
- 1.0 mg/ml CLS-Type I bacterial Collagenase (Worthington Biochemical Corp.,
Freehold, NJ, #LS00 04197).
- 0.375 mg/ml Type IS Soybean Trypsin Inhibitor (Worthington Biochemical Corp.,
Freehold, NJ, #LS00 03571).
- 0.125 mg/ml Type III Elastase (Sigma Chemical, St. Louis, MO, #E-0127).
5. Mixture incubated for 90 minutes at 37°C in a gyratory shaker bath ~120 RPM.
6. Digest poured over a 110 micron stainless steel mesh (Cellector, Bellco Glass). Filtrate discarded and residue washed from filter with 18 ml Dulbecco's Modified Eagle's medium containing 10% (n/n) heat-inactivated calf serum, 2 mM L-glutamine and 100 units/m1 penicillin, 100 mg/ml streptomycin. (Biowhittaker).
7. Tissue + media pipetted into a 20 ml plastic syringe and gently drawn back and forth 10 times through a 12 or 14 gauge stainless steel cannula (Becton-Dickinson). Tissue suspension sieved on 110 micron stainless steel mesh to separate dispersed cells from undigested vessel wall fragments and debris.
8. Filtrate centrifuged in a siliconized conical glass tube at 200 g x 5 min. Supernatant aspirated and pellet resuspended in Dulbecco's Modified Eagle Medium with serum (above) for culture.
Wm. Atkinson/M. Gimbrone
Vascular Pathophysiology Laboratory
Department of Pathology
Brigham & Women's Hospital
Boston, MA 02115
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