TITLE: No Title
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[DESCRIPTION]
<p>CasPASE™ Apoptosis Fluorometric Assay provides a simple method for assaying the activities of various caspases (proteases), a key early indicator of apoptosis in mammalian cells. </p>

[BEFORE_START]
<p><em><strong>Preparation of Kit Reagents  </strong> </em></p>
<p>1. Allow the reagents to thaw into liquid form. Centrifuge the substrate, free dye and the inhibitor vials to collect the reagent solution at the bottom of the vial. Protect from light and humidity. Allow the reagents to reach RT before opening the vial.  </p>
<p>2. Dissolve the supplied DTT in 90µl DI water (final volume 100µl) to give 1M concentration. Store at ‐20°C.</p>

[GUIDELINES]
<p><strong>INTRODUCTION</strong></p>
<p>CasPASE™ Apoptosis Fluorometric Assay provides a simple method for assaying the activities of various caspases (proteases) (Caspase 1‐10 &amp; 13), a key early indicator of apoptosis in mammalian cells. The assay is based on the detection of cleavage of a synthetic substrate, which has 7‐amino‐4‐trifluromethyl coumarin (AFC) at the C‐ terminal. When liberated from the peptide, AFC produces an optical change that can be detected as the fluorescence at 500‐550nm with the use of a fluorometer. The reaction is selectively and irreversibly inhibited by the peptide Z‐VAD‐FMK (fluoromethyl ketone). Comparison of the fluorescence from an iapoptotic sample with a control sample allows one to determine the fold‐increase in the caspase activity. The activity of the caspases can also be quantified by using a standard curve established with the free dye (AFC).</p>
<p> </p>
<p><strong>ITEM(S) SUPPLIED</strong></p>
<table>
<tbody>
<tr>
<td><strong>Description</strong></td>
<td><strong>50 Assay</strong></td>
<td><strong>100 Assay</strong></td>
</tr>
<tr>
<td>CasPASE™ Lysis Buffer</td>
<td>15ml</td>
<td>2 x 15ml</td>
</tr>
<tr>
<td>2X CasPASE™ Assay Buffer</td>
<td>5ml</td>
<td>5ml</td>
</tr>
<tr>
<td>DTT [1M, 100μl]</td>
<td>1 vial</td>
<td>1 vial</td>
</tr>
<tr>
<td>Substrate Solution [1mM]<sup>γ</sup> </td>
<td>0.25ml</td>
<td>2 x 0.25ml</td>
</tr>
<tr>
<td>Free Dye (AFC), [8mM]</td>
<td>0.2ml</td>
<td>0.2ml</td>
</tr>
<tr>
<td>Inhibitor (Z‐VAD‐FMK) [1mM]</td>
<td>0.1ml</td>
<td>2 x 0.1ml</td>
</tr>
</tbody>
</table>
<p><sup>Y</sup> The different substrate solutions supplied with individual kits are as follows:</p>
<p> </p>
<table style="width: 433px;">
<tbody>
<tr>
<td style="width: 79px;"><strong>Cat. #</strong></td>
<td style="width: 141px;"><strong>Description </strong>Substrate supplied</td>
<td style="width: 128px;"><strong>Size</strong></td>
</tr>
<tr>
<td style="width: 79px;">786‐200A 786‐200B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐1, 4, 5 Assay</strong></p>
<p>Ac‐WEHD‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐201A 786‐201B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐2 Assay</strong></p>
<p>Ac‐VDVAD‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐202A 786‐202B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐3, 7,10 Assay </strong></p>
<p>Ac‐DEVD‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐203A 786‐203B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐6 Assay </strong></p>
<p>Ac‐VEID‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐204A 786‐204B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐8 Assay </strong></p>
<p>Ac‐LETD‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐205A 786‐205B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐9 Assay </strong></p>
<p>Ac‐LEHD‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays</td>
</tr>
<tr>
<td style="width: 79px;">786‐206A 786‐206B</td>
<td style="width: 141px;">
<p><strong>CasPASE™ ‐13 Assay</strong>  </p>
<p>Ac‐LEED‐AFC substrate</p>
</td>
<td style="width: 128px;">50 Assays /   100 Assays<strong> </strong></td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>STORAGE CONDITION</strong></p>
<p>The kit is shipped in blue ice. Upon arrival, store all the reagents at ‐20°C. When used properly, the substrates are stable for 6 months and other components for up to 1 year.</p>
<p> </p>
<p><strong>ADDITIONAL ITEMS NEEDED</strong></p>
<p>• Centrifuge</p>
<p>• 96‐well plates or Reaction Tube </p>
<p> </p>
<p><em><strong>Preparation of Kit Reagents  </strong></em></p>
<p>1. Allow the reagents to thaw into liquid form. Centrifuge the substrate, free dye and the inhibitor vials to collect the reagent solution at the bottom of the vial. Protect from light and humidity. Allow the reagents to reach RT before opening the vial.  </p>
<p>2. Dissolve the supplied DTT in 90µl DI water (final volume 100µl) to give 1M concentration. Store at ‐20°C.</p>
<p> </p>
<p><strong>Assay Controls</strong></p>
<p>Prepare a negative control reaction with cells not treated with the apoptosis‐inducing stimulus.</p>
<p> </p>
<p><strong>Assay Protocol</strong> </p>
<p>First read the section “Preparation Before Use”. The assay may be performed in a 96 well microplate or cuvette, using a fluorometer.</p>
<p> </p>
<p>Set up the assay in duplicate and arrange the appropriate blanks and controls, such as a non‐apoptotic cell lysate (negative control).  A blank should be prepared to measure the substrate background and instrument drift.</p>
<p> </p>
<p><strong>Inhibition of Caspase Activity (Optional)</strong></p>
<p>In order to establish non specific protease activity, a control should be run with or without the caspase specific inhibitor (Z‐VAD‐FMK) supplied with the kit.</p>
<p> </p>
<p>1. Reaction tubes should be prepared as described above (e.g., 50μl lysate and 50μl of 2X Assay buffer).</p>
<p>2. Add 1 μl of the Inhibitor (10μM final conc.), mix and incubate the reaction at 20‐ 37°C for 30 minutes to complete the inhibition.</p>
<p>3. Add 5μl of 1mM conjugated substrate (50μM final concentration).</p>
<p>4. Mix the content of the tube and take a reading at zero time point (t = 0).</p>
<p>5. Incubate the assay tubes at 20‐37°C.</p>
<p>6. Measure the reaction every 30‐60 minutes until the sample measurements are complete.</p>
<table style="width: 290px;">
<tbody>
<tr>
<td style="width: 10px;"><strong>Component</strong></td>
<td style="width: 30px;"><strong>Blank</strong></td>
<td style="width: 100px;"><strong>Test Sample</strong></td>
</tr>
<tr>
<td style="width: 10px;">2X Assay Buffer</td>
<td style="width: 30px;">100μl</td>
<td style="width: 100px;">50μl</td>
</tr>
<tr>
<td style="width: 10px;">Test Sample/Lysate</td>
<td style="width: 30px;">---</td>
<td style="width: 100px;">50μl</td>
</tr>
<tr>
<td style="width: 10px;">Inhibitor</td>
<td style="width: 30px;">---</td>
<td style="width: 100px;">0.5μl-1μl </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>FLUORESCENT DETECTION OF THE CASPASE ACTIVITY</strong></p>
<p>Read sample in a fluorometer setting at 360‐390nm EXCITATION and 510‐550nm EMISSION filter. Zero the detection scale using 1x Assay Buffer. Insert the most concentrated solution (4µM AFC) and adjust the setting, e.g. adjust the gain to obtain a signal near the maximum scale. If the blank signal is more than 50% of the full‐scale signal, the substrate may have degraded.</p>
<p> </p>
<p>Comparison of the fluorescence from an induced/apoptotic sample with an uninduced or inhibited control allows one to determine the fold‐increase in protease activity. </p>
<p> </p>
<p><strong>CASPASE ACTIVITY CALCULATION</strong></p>
<p>1. Generate a dye (AFC) calibration curve and determine the slope.</p>
<p>2. Dilute AFC solution in CasPASE™ Lysis Buffer and prepare 0, 10, 20, 40, and 80µM stock solutions.</p>
<p>3. Mix 190μl of CasPASE™ Lysis Buffer with each 10µl of stock solutions, as follows:</p>
<p>          10μl of 0μM AFC + 190μl of Lysis Buffer = 0.0nmole AFC</p>
<p>          10μl of 10μM AFC + 190μl of Lysis Buffer = 0.1nmole AFC (0.5μM)</p>
<p>          10μl of 20μM AFC + 190μl of Lysis Buffer = 0.2nmole AFC (1μM)</p>
<p>          10μl of 40μM AFC + 190μl of Lysis Buffer = 0.4nmole AFC (2μM)</p>
<p>          10μl of 80μM AFC + 190μl of Lysis Buffer = 0.8nmole AFC (4μM)</p>
<p>4. Plot nmole AFC (x‐axis) Vs (FU) fluorescence unit (y‐axis), and determine the slope i.e., FU/nmole AFC.</p>
<p>5. Calculate the rate of increase in fluorescence for each sample as follows:</p>
<p>ΔF / minute = [ΔF<sub> sample</sub> ‐ΔF <sub>blank</sub>] / minute  </p>
<p>(i.e., change in fluorescence over the length of reaction time minus the change in the fluorescence over the same length of reaction for the blank).</p>
<p>6. Calculate unit of caspase activity using the following formula: Units caspase = ΔF / minute x (calibration curve slope) <sup>‐1</sup></p>
<p> </p>
<p><strong>Example </strong></p>
<p>Slope of calibration plot = 2500 (i.e., per nmole of AFC yields 2500 fluorescence units) Sample: in 60 minutes fluorescence change from 0 to 800 FU, i.e., rate of fluorescence increase = 800/60.   Units Caspase in sample = (800/60) x (1/2500) = 0.00533 nmole/minute.</p>
<p> </p>
<p><strong>Citations</strong></p>
<p>1. Lee, R.X. et al (2012) Anticancer Res. 32:3103<br />2. Soong, G. et al (2012) J. Infect. Dis. 205:1571<br />3. Zhang, A. et al (2006) Am. J. Physiol. Renal. Physiol. 291:F1332</p>

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