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[DESCRIPTION]
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<p>This protocol is intended to illustrate the process for testing a particular cell type with the IRDye 800CW EGF for eventual use in vivo. A431 (epithelial carcinoma) cells are used in this example because of their over-expression of EGFR. Media considerations and certain cell characteristics will naturally alter this protocol if a different cell line is used.</p>
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<p> </p>
<p><strong>Developed for:</strong></p>
<p> </p>
<p>Aerius,</p>
<p>Odyssey® Classic,</p>
<p>Odyssey CLx, and</p>
<p>Odyssey Sa</p>
<p>Infrared Imaging Systems</p>
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[GUIDELINES]
<p><strong>I. Required Reagents</strong></p>
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<p><strong>LI-COR Reagents</strong></p>
<ul>
<li>IRDye 800CW EGF (P/N 926-08446)</li>
<li>CellTagTM 700 Stain (LI-COR, P/N 926-41090)</li>
<li>Odyssey® Blocking Buffer (PBS) (LI-COR, P/N 927-40000)</li>
</ul>
<p> </p>
<p><strong>Additional Reagents</strong></p>
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<ul>
<li>1X PBS wash buffer</li>
<li>Tissue culture reagents (serum, DMEM, trypsin, 1X PBS)</li>
<li>100% Tween® 20</li>
<li>37% formaldehyde</li>
<li>10% Triton® X-100</li>
<li>Costar™ 96-well Black Clear-Bottom Plate (Corning)</li>
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<p>SPECIAL NOTE: Serum starvation of the cells is required to obtain maximal response.</p>
<p> </p>
<p><strong>II. Seeding, Stimulation, and Detection with IRDye 800CW EGF</strong></p>
<p> </p>
<p>"See STEPS"</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hatc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hatc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.56.47 AM.png" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hauc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hauc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.57.32 AM.png" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/havc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/havc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.58.18 AM.png" /></p>
<p> </p>
<p><strong>III. Experimental Considerations</strong></p>
<p> </p>
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<p>Proper selection of microplates can significantly affect the results of an analysis, as each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover. Use the general considerations for microplate selection provided below.</p>
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<ul>
<li>On-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear and black-sided plates with clear bottoms. Do not use plates with white wells, since the autofluorescence from the white surface will create significant noise.</li>
<li>On-Cell Western assays require sterile plates for tissue culture growth. The following plates and focus offset settings are recommended by LI-COR Biosciences. Please be aware that manufacturers’ specifications for culture plates are subject to change.</li>
<li>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.</li>
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<td>
<p><strong>Well</strong></p>
<p><strong>Number</strong></p>
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<td>
<p><strong>Well</strong></p>
<p><strong>Bottom</strong></p>
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<td><strong>Manufacturer</strong></td>
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<p><strong>Part</strong></p>
<p><strong>Numbers</strong></p>
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<td>
<p><strong>Odyssey &amp; </strong></p>
<p><strong>Odyssey CLx</strong></p>
<p><strong>Offset</strong></p>
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<td>
<p><strong>Aerius &amp;</strong></p>
<p><strong>Odyssey Sa</strong></p>
<p><strong>Offset</strong></p>
</td>
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<tr>
<td>96</td>
<td>Flat</td>
<td>Nunc</td>
<td>161093, 165305</td>
<td>3.0 mm</td>
<td>3.0 mm</td>
</tr>
<tr>
<td>96</td>
<td>Flat</td>
<td>BD Falcon</td>
<td>353075, 353948</td>
<td>3.0 mm</td>
<td>3.0 mm</td>
</tr>
<tr>
<td>96</td>
<td>Flat</td>
<td>Corning</td>
<td>3603</td>
<td>3.0 mm</td>
<td>3.0 mm</td>
</tr>
<tr>
<td>384</td>
<td>Flat</td>
<td>Nunc</td>
<td>164688, 164730</td>
<td>3.0 mm</td>
<td>3.0 mm</td>
</tr>
<tr>
<td>384</td>
<td>Flat</td>
<td>BD Falcon</td>
<td>353961, 353962</td>
<td>3.0 mm</td>
<td>3.0 mm</td>
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<p> </p>
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<p><strong>Focus Offset Optimization</strong> - Plates deviating from LI-COR recommendations may require lower or higher focus offsets for optimal resolution and detection.</p>
<p> </p>
<table>
<tbody>
<tr>
<td>Instrument</td>
<td>
<p>Focus Offset</p>
<p>Determination (mm)</p>
</td>
</tr>
<tr>
<td>Odyssey Classic &amp; Odyssey CLx</td>
<td>0.5, 1.0, 2.0, 3.0 &amp; 4.0</td>
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<tr>
<td>Odyssey Sa &amp; Aerius</td>
<td>1.7, 2.0, 3.0 &amp; 4.0</td>
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<p> </p>
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<p>Alternatively, consult the plate manufacturer to obtain the measured distance from the skirt to the bottom of the plate. Microplates are required to have a distance of no more than 4.0 mm from the instrument surface to the target detection area of the plate.</p>
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<p><strong>Intensity Setting Optimization</strong> – Intensity settings may need to be optimized depending on results. If signal is weak or saturated, try the suggested intensity settings in the table below to achieve optimal results.</p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p> </p>
<p> </p>
<p> </p>
<p>Instrument</p>
</td>
<td>
<p>Initial</p>
<p>Intensity</p>
<p>Setting</p>
<p>(700/800 nm)</p>
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<td>
<p>Intensity</p>
<p>Settings:</p>
<p>Weak Signal</p>
<p>(700/800 nm)</p>
</td>
<td>
<p>Intensity</p>
<p>Settings:</p>
<p>Saturated Signal</p>
<p>(700/800 nm)</p>
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<tr>
<td>
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<p>Odyssey® Classic</p>
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</td>
<td>
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<p>5 / 5</p>
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</td>
<td>7.5 / 7.5</td>
<td>2.5 / 2.5</td>
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<td>
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<p>Odyssey CLx</p>
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</td>
<td>
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<p>5 / 5</p>
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</td>
<td>7.5 / 7.5</td>
<td>2.5 / 2.5</td>
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<td> </td>
<td>
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<p>AutoScan*</p>
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</td>
<td>-</td>
<td>-</td>
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<td>
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<p>Odyssey Sa</p>
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</td>
<td>7 / 7</td>
<td>8 / 8</td>
<td>4 / 4</td>
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<tr>
<td>
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<p>Aerius</p>
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</td>
<td>7 / 7 </td>
<td>8 / 8</td>
<td>4 / 4<br /><br /></td>
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<p>*The Image Studio AutoScan function for the Odyssey CLx alleviates the need to scan the plate at multiple intensity settings.</p>
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[STEPS]
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