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<p>The In-Cell Western assay is a popular immunoassay for the study of signal transduction, protein expression, and function. A key feature in this assay is its ability to simultaneously measure two targets of interest or normalize the data for well-to-well variation in cell number. LI-COR has developed three types of protocols for normalization.</p>
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<p> If optimal fixation and permeabilization conditions for immunofluorescent staining of your cell line and/or target protein are already known, these conditions may be more appropriate than the fixation protocol described here, and would be an excellent starting point for In-Cell Western assay development. Most fixatives and fixation protocols for immunofluorescent staining may be adapted to the In-Cell Western format.</p>

[GUIDELINES]
<p><strong>I. Introduction</strong></p>
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<p>The In-Cell Western assay is a popular immunoassay for the study of signal transduction, protein expression, and function. A key feature in this assay is its ability to simultaneously measure two targets of interest or normalize the data for well-to-well variation in cell number. LI-COR has developed three types of protocols for normalization.</p>
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<p>The first method uses primary and secondary antibodies to detect two distinct targets. For example, phospho-ERK can be detected using a specific primary antibody and a secondary antibody labeled with IRDye 800CW infrared dye. In a multiplex assay, a primary antibody against total ERK (or against a housekeeping protein or other target) can be detected with a secondary antibody labeled with a spectrally-distinct IRDye fluorophore1, such as IRDye 680RD. The protocol has been widely used in scientific literature<sup>1-5</sup>.</p>
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<p>LI-COR also offers a one-step solution for In-Cell Western assay normalization using CellTag™ 700 Stain (LI-COR P/N 926-41090). CellTag 700 Stain allows for normalization across the same range of cell densities as Sapphire700TM and DRAQ5® Stains combined and enables accurate measurement of target protein levels when combined with an IRDye 800CW secondary antibody. Please visit http://www.licor.com/bio for more detailed information regarding CellTag 700 Stain.</p>
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<p>The protocol described here is cost-effective, and provides quantification over a wide linear range in a manner that does not use DNA staining and is not affected by changes in nuclear DNA. It was first described by Hoffman and colleagues<sup>6</sup>. This method uses infrared reactive dyes to covalently label cellular proteins on lysine residues. IRDye 800CW or IRDye 700DX N-hydroxysuccinimidyl ester (NHS) reactive dyes can couple to free amine groups on lysine residues and form a stable conjugate. Because the cells are fixed, the reactive dye has access to both cell surface and internal lysine residues, which greatly increases the extent of labeling. These dyes are available in several formats:</p>
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<p>As a component of IRDye Infrared Dye labeling kits. These kits contain other components typically used to label antibodies and other proteins, which are not used in this cell labeling protocol.</p>
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           - IRDye 700DX Protein Labeling Kit, High Molecular Weight (P/N 928-38046)<br />           - IRDye 800CW Protein Labeling Kit, High Molecular Weight (P/N 928-38040)<br />
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<p>This method adds only two brief steps to the protocol and provides several advantages over previous methods:</p>
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<p><em>Extreme sensitivity.</em> The lower-limit detection is approximately 200 cells per well.</p>
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<p><em>Quantitative accuracy.</em> Wide linear range of signal extends from 200 to 200,000 cells in</p>
<p>our experiments (Fig. 1).</p>
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<p><em>Cost-effectiveness.</em> Because highly-diluted dye solutions are used, a 0.5 mg vial of IRDye 700DX reactive dye can label 50 plates of cells; a 0.5 mg vial of IRDye 800CW is sufficient to label 500 plates (Figure 2).</p>
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<div class="column"><strong>II. Suggested Materials</strong></div>
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<p>IRDye 800CW OR IRDye 700DX NHS ester reactive dye</p>
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           - IRDye 800CW NHS ester, 0.5 mg (P/N 929-70020) or 5 mg (929-70021)<br />           - IRDye 700DX NHS ester, 0.5 mg (P/N 929-70010) or 5 mg (929-70011)<br />
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<p>Dry (anhydrous grade) dimethyl sulfoxide (DMSO)</p>
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<p>Odyssey® Blocking Buffer (PBS) (P/N 927-40000)</p>
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<p>1X PBS wash buffer</p>
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<p>Tissue culture reagents (serum, DMEM, trypsin, etc.)</p>
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<p>Clear or black 96-well microplate, with clear bottom</p>
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<p>37% formaldehyde</p>
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<p>20% Tween® 20</p>
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<p>10% Triton® X-100</p>
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<p><strong>III. Protocol</strong></p>
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<p>Treat cells as desired with drug, stimulant, etc. Detailed In-Cell Western protocols for certain cell lines and target proteins may be downloaded at <a href="http://www.licor.com/icwprotocols" target="_blank" rel="noopener noreferrer">www.licor.com/icwprotocols</a></p>
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<p><em>IMPORTANT NOTE: If optimal fixation and permeabilization conditions for immunofluorescent staining of your cell line and/or target protein are already known, these conditions may be more appropriate than the fixation protocol described here, and would be an excellent starting point for In-Cell Western assay development. Most fixatives and fixation protocols for immunofluorescent staining may be adapted to the In-Cell Western format.</em></p>
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<p><em>NOTE: If cells are loosely attached to plate, centrifuge plate at ~350 x g for 5-10 minutes during the last 10-15 minutes of this incubation. This will help prevent cell loss.</em></p>
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<p>See 'STEPS'</p>
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<p><strong>References</strong></p>
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<p>Chen, H et al. A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Analyt Biochem 338: 136-142 (2005).</p>
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<p>Wong, SKF. A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors. Analyt Biochem 333: 265-272 (2004).</p>
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<p>Selkirk, JV et al. A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation. Biomolecular Screening 11: 351-358 (2006).</p>
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<p>Kumar, N et al. Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpresssing mammary epithelial cells. Mol Pharmacol 73:1668-1678 (2008).</p>
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<p>Hannoush, RN. Kinetics of Wnt-driven b-catenin stabilization revealed by quantitative and temporal imaging. PLoS ONE 3(10):e3498 (2008).</p>
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<p>Hoffman, G et al. A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, American Society for Cell Biology Annual Meeting (2008).</p>
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<p>Hoffman, GR et al. A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In-Cell Western technique. Assay Drug Dev Technol. doi:10.1089/adt.2009.0213 (2010).</p>
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[STEPS]
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