Core Facilities
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,
Indiana, #81-001-3).
- 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
(617) 732-5901
Vascular Pathophysiology Laboratory
Department of Pathology
Brigham & Women's Hospital
Boston, MA 02115
(4/1981) (5/19/00) |
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