Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Summary

Multiplex assays can provide beneficial information for basic cellular mechanisms and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

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.

Introduction

Diets rich in saturated fatty acids such as palmitic acid (PA) have been linked to obesity and other comorbidities, including cardiovascular disease and diabetes^1,2. High fat diets have also been shown to increase oxidative stress, apoptosis, and neuronal degeneration in the hypothalamus, an important regulator of appetite and energy expenditure^3-7. Understanding the mechanism through which high fat diet exposure induces hypothalamic dysregulation is thus important for development of pharmacological treatments for obesity. However, the cellular mechanisms through which dietary fat affects neuronal function remain unclear. A ....

Protocol

1. Plating and Maintenance of Cell Culture

1. Warm Dulbecco's Modified Eagle Medium (DMEM) culture media (DMEM + 10% FBS + 1% Penicillin/Streptomycin/Neomycin) to 37 °C.
2. Obtain stock of frozen A12 cells from -80 °C.
3. Rapidly thaw cells in 37 °C water bath; once thawed, gently pipette cells to transfer to a 75 cm² vented culture flask and add 10 ml of culture media.
4. Incubate flask overnight in 5% CO₂ incubator at 37 °C. Aspirate media after 24 hr and replace with fresh media. Continue growing cells until 70-80% confluence is obtained (2-3 days growth).

2....

Results

The protocol above describes results in multiplexing two separate assays to determine mechanisms of cell death. Figure 1 shows an overview of the protocol to determine cell viability and caspase-3/7 activity. Caspase activity was significantly increased in cells challenged with PA after 2 hr incubation (Figure 2A and Table 1). Loss of cell membrane integrity is a morphological change associated with the induction of apoptosis, which is apparent in cells after 2 hr exposu.......

Discussion

The multiplex assay is a well-accepted technique that has been used by scientists in numerous applications such as PCR microarrays, immunodetection, and other protein-based detection methods^13,14. Recently, multiplexing assays have become increasingly utilized in in vitro plate-based experiments and have been validated as an accurate method of assessing cytotoxicity and viability¹². In the above protocol, we demonstrate the effectiveness of multiplexing fluorescent and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adult Mouse Hypothalamus Cell Line mHypoA-1/2	Cellutions Biosystems Inc.	CLU172
Dulbecco’s Modified Eagle’s Medium	Invitrogen	10313-039
Fetal Bovine Serum	PAA Labs	A15-751
Penicillin/Streptomycin	Invitrogen	15070-063
Palmitic Acid	Sigma-Aldrich	P0500
Dimethy Sulfoxide	Sigma-Aldrich	D2650
PrestoBlue Cell Viability Reagent	Invitrogen	A13262
Caspase-Glo 3/7 Assay Systems	Promega	G8091
96 W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile	Thermo Fisher Scientific	165305
SpectraMax M5 Multi-Mode Microplate	Molecular Devices