Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model |
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Authors: | Tammy A Butterick Cayla M Duffy Rachel E Lee Charles J Billington Catherine M Kotz Joshua P Nixon |
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Institution: | 1.Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System;2.Department of Food Science and Nutrition, University of Minnesota;3.Department of Integrative Biology and Physiology, University of Minnesota;4.Department of Medicine, University of Minnesota Medical School, University of Minnesota |
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Abstract: | The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function. |
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Keywords: | Neuroscience Issue 86 apoptosis obesity caspase resazurin DEVD palmitic acid hypothalamic model |
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