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[DESCRIPTION]
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<p><strong>Developed for:</strong></p>
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<p>Aerius,<br /> Odyssey® Classic,</p>
<p>Odyssey CLx, and</p>
<p>Odyssey Sa<br /> Infrared Imaging Systems</p>
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[GUIDELINES]
<p><strong>Required Reagents</strong></p>
<p><strong>LI-COR Reagents</strong></p>
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<ul>
<li>
<p>IRDye® 800CW Goat anti-Rabbit Secondary Antibody (LI-COR P/N 925-32210 or 926-32210)</p>
</li>
<li>
<p>IRDye 680RD Goat anti-Mouse Secondary Antibody (LI-COR P/N 925-68070 or 926-68070)</p>
</li>
<li>
<p>Odyssey® Blocking Buffer (LI-COR P/N 927-40000 or 927-50000)</p>
</li>
<li>
<p>Large Western Incubation Box (LI-COR P/N 929-97301)</p>
</li>
</ul>
<p> </p>
<p><strong>Additional Reagents</strong></p>
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<li>1X PBS wash buffer</li>
<li>Tissue culture reagents (RPMI 1640, fetal bovine serum, etc.)</li>
<li>Clear or black 96-well or 384-well microplates (See IX. Experimental Considerations)</li>
<li>Jurkat cells (ATCC®, P/N TIB-152TM)</li>
<li>THP-1 monocytes (ATCC, P/N TIB-202TM)</li>
<li>K-562 lymphocytes (ATCC, P/N CCL-243TM)</li>
<li>Concentrated Prefer (5X) (Anatech LTD, P/N 411)</li>
<li>TO-PRO®-3 (Molecular Probes, P/N T-3605)</li>
<li>PMA (phorbol 12-myristate 13-acetate) (Sigma®, P/N P1585)</li>
<li>DMSO (dimethyl sulfoxide) (Sigma, P/N D8418)</li>
<li>ERK rabbit antibody (Santa Cruz, P/N sc-94)</li>
<li>pERK mouse antibody (Cell Signaling Technology, P/N 9106)</li>
</ul>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hanc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hanc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.00.41 AM.png" /></p>
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<p><strong>Figure 1.</strong> Plate layout of experiment. Round bottom 96-well plate: ~200,000 Jurkat cells per well (C1-E12) in triplicate samples. (Data shown in Figure 2)</p>
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<p><strong>Experimental Considerations</strong></p>
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<p>Proper selection of microplates can significantly affect results, 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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<li>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. <strong>Do not use plates with white walls, since autofluorescence from the white surface will create significant noise.</strong></li>
<li>
<p>Both the flat and round-bottom plates show some plate autofluorescence; however, it is relatively small compared to the actual signal. When using the recommended BD Bioscience round-bottom plate, the recommended focus offset is 3.0 to 3.5 mm. The recommended focus offset for the Nunc round-bottom plate is 3.5 to 3.95 mm.</p>
</li>
<li>
<p><strong>Focus Offset Optimization</strong> - If plates other than those recommended are used, the focus off- set can be determined empirically by scanning a plate containing experimental and control samples using the following focus offset settings.</p>
</li>
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<td><strong>Instrument</strong></td>
<td>
<p><strong>Focus Offset</strong></p>
<p><strong>Determination (mm)</strong></p>
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<p>Odyssey Classic &amp; Odyssey CLx</p>
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<p>1.0, 1.5, 2.0, 2.5, 3.0, 3.5, &amp; 4.0</p>
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<p>Odyssey Sa &amp; Aerius</p>
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<p>1.5, 2.0, 2.5, 3.0, &amp; 3.95</p>
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<p> </p>
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<p>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, consult the plate manufacturer for the recommended measured distance from the skirt bottom to the bottom of the plate.</p>
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<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>
<table>
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<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>
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<td>
<p>Intensity</p>
<p>Settings:</p>
<p>Saturated Signal</p>
<p>(700/800 nm)</p>
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<p>Odyssey® Classic</p>
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<p>5 / 5</p>
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<td>7.5 / 7.5</td>
<td>2.5 / 2.5</td>
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<p>Odyssey CLx</p>
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<p>5 / 5</p>
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<td>7.5 / 7.5</td>
<td>2.5 / 2.5</td>
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<td> </td>
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<p>AutoScan*</p>
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</td>
<td>-</td>
<td>-</td>
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<p>Odyssey Sa</p>
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<td>7 / 7</td>
<td>8 / 8</td>
<td>4 / 4</td>
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<p>Aerius</p>
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<td>7 / 7 </td>
<td>8 / 8</td>
<td>4 / 4</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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<ul>
<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>
<li>Establish the specificity of your primary antibody by screening lysates through Western blotting. 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.</li>
</ul>
<p> </p>
<p><strong> Experimental Results</strong></p>
<p>                                                          phorbol 12-myristate 13-acetate (ng/mL)</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hapc2w6.png" width="532" height="272" data-src="https://s3.amazonaws.com/pr-journal/hapc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.10.40 AM.png" /></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/haqc2w6.png" width="529" height="463" data-src="https://s3.amazonaws.com/pr-journal/haqc2w6.png" data-ofn="Screen Shot 2016-12-15 at 10.11.37 AM.png" /></p>
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