Induction of CYP1A2, 2B6, and 3A In Fresh Human Hepatocytes<strong>This assay is used to assess the potential of a test compound to induce CYP1A2, CYP2B6, and CYP3A in fresh human hepatocytes</strong>

Catalog number EA451

<strong>Required from Customer</strong>
<ul>
 	<li>Either a minimum of 600 µL of test compound at 10 mM in DMSO, or 10 mg of powder</li>
 	<li>The molecular mass (exact mass) of test compound and its salt form</li>
 	<li>The MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.</li>
</ul>
<strong>Deliverables</strong>
<ul>
 	<li>Table of MTS absorbance values (an index of cell viability) at each concentration of test compound and in controls</li>
 	<li>Tables of CYP1A2, CYP2B6, and CYP3A mRNA expression for each treatment group</li>
 	<li>Percentage of induction by each concentration of the test compound relative to the solvent and positive controls</li>
</ul>
<strong>Substrate</strong>
<ul>
 	<li>Test compound dissolved in DMSO, acetonitrile, or methanol</li>
</ul>
<strong>Assay System</strong>
<ul>
 	<li>Plated fresh human hepatocytes from 3 individual donors</li>
</ul>
<strong>Assay Conditions</strong>
<ul>
 	<li>Three concentrations of test compound, typically 1, 10 and 100 µM</li>
 	<li>Triplicate incubations (N=3) at each concentration of test compound, positive controls, and negative controls</li>
 	<li>Positive control compounds are known inducers of each CYP</li>
 	<li>Negative control is the vehicle only</li>
 	<li>Incubate hepatocytes with test compound, vehicle, and positive controls in parallel wells for 72 hours at 37°C, replacing with fresh compound-containing medium daily</li>
 	<li>Determine mRNA expression of each CYP in test compound, vehicle and positive control wells</li>
</ul>
<strong>Assay QC</strong>
<ul>
 	<li>In wells treated with positive control, CYP activity/mRNA expression &gt;2-fold the activity in negative control</li>
 	<li>Determine cell viability in the same wells used to determine CYP activity, using conversion of a tetrazolium salt (MTS) to the corresponding formazan product</li>
</ul>
<strong>Notes</strong>
<ol>
 	<li>CYP-specific inducers as positive controls</li>
</ol>
<table>
<tbody>
<tr>
<td width="75"><strong>CYP
Isoform</strong></td>
<td width="175"><strong>Positive Control
(CYP Inducer)</strong></td>
</tr>
<tr>
<td>1A2</td>
<td>Omeprazole, 50 µM</td>
<td></td>
</tr>
<tr>
<td>2B6</td>
<td>Phenobarbital, 1000 µM</td>
<td></td>
</tr>
<tr>
<td>3A</td>
<td>Rifampicin, 50 µM</td>
<td></td>
</tr>
</tbody>
</table>
<ol>
 	<li>Cell viability is determined by measuring the concentration of formazan dye converted from MTS by metabolically active hepatocytes based on absorbance at 492 nm.</li>
</ol>