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Document Type

Thesis - University Access Only

Award Date

1992

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

David J. Hurley

Abstract

The physiological role of ions, specifically calcium, in living cells has been studied by use of fluorescent probes. A widely used fluorescent probe sensitive to calcium is fura-2. The cell permeant form of fura-2 is an acetoxy methyl ester, fura-2 AM. A common method for loading fura-2 AM into cells employs the detergent Pluronic F127 (F127) and fetal bovine serum (FBS). The FBS aids in localizing fura-2 AM to the cell and the F127 facilitates movement of the dye across plasma membrane. This method is complicated by the membrane destabilization induced by the detergent and calcium redistribution caused by FBS. In order to improve the physiological conditions for intracellular studies we developed a detergent-free (DF) fura-2 AM loading medium. The DF medium is composed of a lipid solution to localize and aid in transmembrane transport of the fura-2 AM. Comparing cells loaded using the F127 loading method and the DF method, DF loaded cells demonstrated larger fura-2 responses upon treatment with stimulant(s). In addition, OF loaded cells retained greater viability than F127 loaded cells, over the course of the experiments. Our studies show that the DF loading method allows for a more native cellular physiology. DF loaded cells demonstrated four advantages over the F127 loading method: 1) Higher receptor-lectin binding capacity, 2) Essentially native membrane packing, 3) Morphology analogous to untreated cells, and 4) Greater esterase activity.

Library of Congress Subject Headings

Calcium -- Physiological effect
Fluorescence
Lymphocytes
Mitogens

Format

application/pdf

Publisher

South Dakota State University

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