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ISOLATION OF PARIETAL CELLS FROM GLUTARALDEHYDE-FIXED RABBIT STOMACH

SIN HANG LEE 1

1 Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510

A method is introduced to isolate and purify parietal cells from the rabbit stomach which has been briefly perfused with 1% glutaraldehyde via the celiac artery. The fixed mucosa is initially digested with 1% trypsin and subsequently with 1% collagenase solution. The parietal cells are more resistant to the effects of these proteolytic enzymes than the other cell types in the gastric mucosa and can be recovered and purified by centrifugation in a discontinuous sucrose gradient. Glutaraldehyde fixation reduces the tendency of cell clumping and, therefore, facilitates isolation of the separated cells. The final yield from each adult rabbit stomach varies from 0.1 to 0.4 ml packed parietal cells of about 97% purity determined by nuclear counts. The nuclei and mitochondria of most of the isolated cells appear to be intact on electron micrographs, but the cell membrane is focally disrupted; the nuclear deoxyribonucleic acid (DNA) can be stained with Feulgen technique and extracted with perchloric acid for quantitative determination. Biochemical studies on incorporation of thymidine-3H into DNA of the parietal cells have been carried out in normal young adult rabbits and in rabbits under chronic histamine stimulation. The results show that there is no significant incorporation of thymidine-3H by mature parietal cells in any of the two groups of animals, although the whole mucosa DNA hydrolysates always exhibit a high specific activity.

Submitted on February 24, 1972


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