The work described here was funded by the U S Department of Veterans Affairs Biomedical Laboratory Research and Development BX001686-1A1 and VA Rehabilitation Research and Development.

1. Plating and Maintenance of Cell Culture
<ol>
	<li>Warm Dulbecco&#39;s Modified Eagle Medium (DMEM) culture media (DMEM + 10% FBS + 1% Penicillin/Streptomycin/Neomycin) to 37 &#176;C.</li>
	<li>Obtain stock of frozen A12 cells from -80 &#176;C.</li>
	<li>Rapidly thaw cells in 37 &#176;C water bath; once thawed, gently pipette cells to transfer to a 75 cm2 vented culture flask and add 10 ml of culture media.</li>
	<li>Incubate flask overnight in 5% CO2 incubator at 37 &#176;C. Aspirate media afte...

<ol>
	<li>Akil, L., Ahmad, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+obesity+and+cardiovascular+diseases+in+four+southern+states+and+Colorado.">Relationships between obesity and cardiovascular diseases in four southern states and Colorado.</a> J. Health Care Poor Underserved. 22, 61-72 (2011).</li><li>Posey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+proinflammatory+lipid+accumulation,+inflammation,+and+insulin+resistance+in+rats+fed+a+high-fat+diet.">Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet.</a> Am. J. Physiol. Endocrinol. Metab. 296, 1003-1012 (2009).</li><li>Moraes, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fat+diet+induces+apoptosis+of+hypothalamic+neurons.">High-fat diet induces apoptosis of hypothalamic neurons.</a> PloS one. 4, (2009).</li><li>Mayer, C. M., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+attenuates+insulin+signaling+and+induces+endoplasmic+reticulum+stress+and+apoptosis+in+hypothalamic+neurons:+rescue+of+resistance+and+apoptosis+through+adenosine+5'+monophosphate-activated+protein+kinase+activation.">Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation.</a> Endocrinology. 151, 576-585 (2010).</li><li>Benoit, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+mediates+hypothalamic+insulin+resistance+by+altering+PKC-theta+subcellular+localization+in+rodents.">Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.</a> J. Clin. Invest. 119, 2577-2589 (2009).</li><li>Thaler, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+is+associated+with+hypothalamic+injury+in+rodents+and+humans.">Obesity is associated with hypothalamic injury in rodents and humans.</a> J. Clin. Invest. 122, 153-162 (2012).</li><li>Williams, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+dysfunction+in+obesity.">Hypothalamic dysfunction in obesity.</a> Proc. Nutr. Soc. 71, 521-533 (2012).</li><li>Belsham, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+phenotypic+array+of+hypothalamic+neuronal+cell+models+to+study+complex+neuroendocrine+disorders.">Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders.</a> Endocrinology. 145, 393-400 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay.">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl. Biochem. Biotechnol. 162, 1996-2007 (2010).</li><li>Butterick, T. A., Nixon, J. P., Billington, C. J., Kotz, C. 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The authors have no conflicts to disclose.

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 methods13,14. 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...

Diets rich in saturated fatty acids such as palmitic acid (PA) have been linked to obesity and other comorbidities, including cardiovascular disease and diabetes1,2. 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 expenditure3-7. 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 better understanding of how fats such as PA might trigger onset of hypothalamic apoptotic pathways is a necessary first step toward this aim. The goal of this article is to describe a multiplex assay for in vitro testing of neuronal response to PA exposure, developed for use in studies of hypothalamic neurodegeneration. We provide a detailed description of an in vitro 96-well format multiplex assay for measuring caspase 3/7 activity per total number of cells in a differentiated immortalized adult mouse hypothalamic cell line (designated A12) after oxidative challenge with PA8.
Briefly, cell viability is determined following PA challenge via a resazurin based assay. Resazurin is a cell permeable compound that undergoes enzymatic reduction in metabolically active cells, a process thought to occur via the mitochondria9. Viable cells continuously convert resazurin to resorufin, producing a fluorescent signal proportional to the number of viable cells. Caspase-3/7 activity is then analyzed using a DEVD-based lumogenic assay. DEVD is an amino acid sequence (Asp-Glu-Val-Asp) cleaved by caspase-3. When this sequence is coupled to a lumogenic substance, upon activation of intracellular caspase-3/7 and subsequent cleavage of DEVD substrate, the luminescent product is released. This reaction is proportional to caspase activity and thus to the induction of apoptosis. As dead cells cannot produce caspase, caspase-3/7 activity is by nature transient; therefore analysis should be completed between 30 min to 4 hr post-challenge, depending on effectiveness of cell stressor. Cell viability is inversely proportional to caspase-3/7 activity and can be used to determine mechanisms of cell death. For example, this method has previously been used to show that pretreatment with the peptide hormone orexin reduces apoptosis in hypothalamic cells challenged with hydrogen peroxide, suggesting that mechanisms affected by this treatment are important in protecting against oxidative damage10. It is important to note these assays are cell line- and tissue-dependent, as they rely upon mitochondrial activity to reduce assay reagents. This protocol has been optimized for adult mouse hypothalamic (A12) cells; however, methods described may be altered to fit within the scope of similar research.
Multiplexing assays from a single culture well provides an advantage over the traditional method of performing individual assays for several reasons. In addition to saving time, cell samples, and culture reagents, multiplexing assays can also provide knowledge of cell survival and death, provide internal controls, and eliminate the need for repetitive experiments11,12.
Alternative methods have relied upon Western blots or ELISAs, which are reliable assays, but are expensive and time consuming (1-2 days), especially when using a 96-well format. Excluding the time it takes to culture cells for the multiplexing assay, the total time is less than 3 hr. While this protocol has been optimized for use with A12 cells, it may be altered for use in other models while keeping in mind factors that may influence cell integrity. Determining if this protocol would work for a series of experiments depends upon on the number of samples or experimental conditions and on planned downstream experiments.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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.

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...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

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

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;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.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

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Research

JoVE Journal

Neuroscience

14.9K Görüntüleme.  Minneapolis Veterans Affairs Health Care System.  Multiplex deneyleri temel hücresel mekanizmaların için yararlı bilgi sağlamak ve reaktifler ve gereksiz tekrarlı deneyler atık ortadan kaldırabilir. Biz, palmitik asit ile oksidatif karşılaştırmadan sonra, bir in vitro modelde hipotalamik hücre yaşayabilirliğini belirlemek için, flüoresan ve ışıldayan dayalı yöntemler kullanılarak, burada çok katlı bir kaspaz-3/7 aktivite deneyi tarif eder. 