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J Vis Exp. 2014 Apr 16;(86). doi: 10.3791/51305.

Use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model.

Author information

  • 1Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System; Department of Food Science and Nutrition, University of Minnesota; butte017@umn.edu.
  • 2Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System; Department of Food Science and Nutrition, University of Minnesota.
  • 3Department of Integrative Biology and Physiology, University of Minnesota.
  • 4Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System; Department of Food Science and Nutrition, University of Minnesota; Department of Medicine, University of Minnesota Medical School, University of Minnesota.

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.

PMID:
24797379
PMCID:
PMC4172056
DOI:
10.3791/51305
[PubMed - indexed for MEDLINE]
Free PMC Article
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