TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p>This is a complete apoptosis assay example. It details the seeding, induction, and detection of the HeLa cellular response to Anisomycin treatment.</p>
<p> </p>
<p><strong>Developed for:</strong></p>
<p> </p>
<p>Aerius,<br /> Odyssey® Classic,</p>
<p>Odyssey CLx, and</p>
<p>Odyssey Sa<br /> Infrared Imaging Systems</p>
<p> </p>
<p><strong>Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model.</strong></p>
</div>
</div>
</div>

[GUIDELINES]
<p><strong>I. Required Reagents</strong></p>
<p><strong>LI-COR Reagents</strong></p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>IRDye® 800CW Goat anti-Mouse Secondary Antibody (LI-COR P/N 925-32210 or 926-32210)</p>
</li>
<li>
<p>IRDye 680RD Goat anti-Rabbit Secondary Antibody (LI-COR P/N 925-668071 or 926-68071)</p>
</li>
<li>
<p>Odyssey® Blocking Buffer (LI-COR P/N 927-40000 or 927-50000)</p>
</li>
</ul>
<p> </p>
<p><strong>Additional Reagents</strong></p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>1X PBS wash buffer</p>
</li>
<li>
<p>Tissue culture reagents (serum, DMEM, trypsin, 1X PBS)(<a href="https://www.protocols.io/view/in-cell-western-assay-hela-cellular-response-to-an-gwibxce?step=9" target="_blank" rel="noopener noreferrer">Step 9</a>)</p>
</li>
<li>
<p>20% Tween® 20</p>
</li>
<li>
<p>Anisomycin (Sigma®, P/N A9789)</p>
</li>
<li>
<p>Trypsin-EDTA Solution (1X) (Sigma, P/N T-3924)</p>
</li>
<li>
<p>37% formaldehyde</p>
</li>
<li>
<p>10% Triton® X-100</p>
</li>
<li>
<p>Clear or black 96-well or 384-well microplates (See IX. Experimental Considerations)</p>
</li>
</ul>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column"><em>NOTE: When using primary antibodies discussed in this protocol, either blocking buffer will provide low background. This may not hold true for other primary antibodies.</em></div>
<div class="column">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<ul>
<li>Primary antibodies:</li>
</ul>
           –  Cleaved Caspase-3 (Cell Signaling Technologies, P/N 9661)<br />
<p>           –  β-Tubulin (D-10), [Santa Cruz, P/N SC-5274] or Anti-β-Tubulin, clone AA2,</p>
<p>               [Upstate Group Inc., P/N 05-661]</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p><strong>IX. Experimental Considerations</strong></p>
<p> </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>Proper selection of microplates can significantly affect the results, as each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover. Use the following general considerations for microplate selection.</p>
<p> </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>In-Cell Western analyses use detection at the well surface with minimal liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear as well as black-sided plates with clear bottoms. Do not use plates with white walls, since autofluorescence from the white surface will create significant noise.</p>
</li>
<li>
<p>In-Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:</p>
</li>
</ul>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>                     96-well format, Eppendorf (P/Ns 0030730119 or 0030741030)</p>
<p>                     96-well, clear, Nunc® (P/Ns 167008, 161093)<br />                     96-well, clear, FalconTM (P/Ns 353075, 353948)<br />                     96-well, black with clear bottom, Nunc (P/N 165305)</p>
<p>                     96-well format, Corning (P/N 3603)</p>
<p> </p>
<p>                     384-well, clear, Nunc (P/Ns 164688, 164730)</p>
<p>                     384-well, clear, Falcon (P/Ns 353229, 353289)</p>
<p>                     384-well, black with clear bottom, Nunc (P/N 142761)</p>
<p>                     384-well format, Corning (P/N 3712)</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<p> </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<ul>
<li><strong>Focus Offset Optimization</strong> – If plates other than those recommended are used, the focus offset can be determined empirically by scanning a plate containing experimental and control samples using the following focus offset settings.</li>
<li><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ys9esfe.png" width="548" height="127" data-src="https://s3.amazonaws.com/pr-journal/ys9esfe.png" data-ofn="Screen Shot 2018-05-29 at 2.21.52 PM.png" /></li>
</ul>
<p><br />Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise ratio for experiments. The actual minimum and maximum focus offset will vary with each instrument. Alternatively, the plate manufacturer can be consulted to obtain the measured distance from the skirt bottom to the bottom of the plate. </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p> </p>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<ul>
<li>All Aerius and Odyssey® Imaging systems (excluding Odyssey Fc) require microplates that have a maximum 4.0 mm distance from the base of the microplate to the target detection area of the plate (actual maximum focus offset varies with each Aerius and Odyssey Sa instrument and is found by choosing Settings &gt; System Administration, then clicking Scanner Information). When using plates specified for In-Cell Western assays, the recommended focus offset is 3.5 mm or higher.</li>
<li><strong>Intensity Setting Optimization -</strong></li>
</ul>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<p><br /> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ytbesfe.png" width="621" height="298" data-src="https://s3.amazonaws.com/pr-journal/ytaesfe.png" data-ofn="Screen Shot 2018-05-29 at 2.23.51 PM.png" /></p>
<p> </p>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at 4 °C.</p>
</li>
<li>
<p>Establish the specificity of your primary antibody by screening lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific binding is present, choose alternative primary antibodies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results.</p>
</li>
</ul>
<p><strong>X. Experimental Results</strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g9tc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9tc2w6.png" data-ofn="Screen Shot 2016-12-14 at 10.40.37 AM.png" /></p>
<p> </p>
<div class="page" title="Page 8">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>Induction of apoptosis in HeLa cells was achieved with increasing concentrations of anisomycin. An increase in Cleaved Caspase-3, a cleaved by-product indicative of apoptosis, is illustrated in the graph. The ultimate result of apoptosis induction is cell death. This can clearly be seen at high concentrations of anisomycin (5 - 40 μM) in this example. The reduction in cell number per well is taken into account when normalizing with another antibody or a DNA stain. In an assay such as this, normalization is very important. See <a href="http://www.licor.com/ICWnormalization" target="_blank" rel="noopener noreferrer">www.licor.com/ICWnormalization</a> for more information on normalization options for In-Cell Western assays<br />.</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.