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<p>This protocol describes a cell viability assay that uses near-infrared fluorescent detection. Sapphire700 Stain is used to determine cell viability by assessing cell membrane integrity, and the assay is imaged with the Odyssey CLx Imaging System.</p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
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<p>Cell viability can be assessed based on various cellular features and mechanisms. These include cell membrane integrity (detected by cell impermeable dyes or leakage of intracellular lactate dehydrogenase (LDH) activity), monitoring of ATP with bioluminescence assays, determining esterase activity with Calcein-AM or Fluorescein-DA, measuring cellular Redox status with MTT, MTS, WST, or XTT, and detecting the mitochondrial membrane potential with JC-1. Various cell viability assays have been developed for plate readers (monitoring absorbance and luminescence), flow cytometry, and image cytometry (e.g. NucleoCounter® NC-3000TM from ChemoMetec); however, none of these assays have been optimized for near-infrared detection with the Odyssey Imaging System.</p>
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<p>This protocol describes a cell viability assay that uses near-infrared fluorescent detection. Sapphire700 Stain is used to determine cell viability by assessing cell membrane integrity, and the assay is imaged with the Odyssey CLx Imaging System.</p>
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<p>Sapphire700 Stain is cell impermeable and non-fluorescent in healthy, intact cells.</p>
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<p>When the cell membrane is damaged, the stain binds to intracellular proteins and becomes fluorescent.</p>
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<p>Fluorescence intensity in the 700 nm channel is correlated to the number of cells with compromised membranes.</p>
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<p>Convenient, ‘mix-and-read’ homogenous assay requires no washing or reagent transfer steps (shown in Figure 1).</p>
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<div class="column">This assay measures the total fluorescence in each microplate well, but does not image or count individual cells.</div>
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<div class="column">For this technical note, A431, Jurkat, and RAW264.7 cells were evaluated. Other cell lines may require optimization. Cell death was induced by applying different concentrations of Stauro- sporine (STS), Camptothecin (CPT) or Saponin.</div>
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<li>Saponins are natural surfactants or detergents, found in many plants, that are used to permeabilize or lyse cells.</li>
<li>CPT, a cytotoxic quinolone alkaloid extracted from Camptotheca acuminate, is a potent inhibitor of topoisomerase I, an enzyme required for DNA synthesis. CPT induces apoptosis in a dose-dependent manner in vitro and is routinely used as a general method for inducing apoptosis<sup>1,2</sup>.</li>
<li>STS is an alkaloid originally isolated from bacterium Streptomyces staurosporeus. STS is an inhibitor of phospholipid/Ca2+ dependent protein kinase (Protein Kinase C; PKC), and prevents binding of ATP to the kinase. There are multiple ways in which STS induces apoptosis. One way is by activating caspase-3. STS is used to induce apoptosis in many mammalian cell types<sup>3</sup>.</li>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hbzc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hbzc2w6.png" data-ofn="Screen Shot 2016-12-16 at 10.58.24 AM.png" /></p>
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<p>I<strong>I. Materials</strong></p>
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<p><strong>LI-COR Reagents</strong></p>
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<li>Sapphire700 Stain (LI-COR, P/N 928-40022)</li>
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<p><strong>Additional Materials</strong></p>
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<p>75-cm<sup>2</sup> cell culture flask (Corning P/N 430641)</p>
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<p>96-well plate, flat bottom, tissue culture treated, black wall with clear bottom (Costar, P/N 3904)</p>
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<p>RAW264.7 cells (ATCC® TIB-71TM)</p>
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<p>A431 cells (ATCC® CRL-1555TM)</p>
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<p>Jurkat cells, Clone E6-1 (ATCC® TIB-152TM)</p>
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<p>Fetal Bovine Serum (FBS) (ATCC, P/N 30-2020)</p>
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<p>Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, P/N D5796)</p>
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<p>RPMI-1640 (Sigma, P/N R5886)</p>
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<p>0.05% Trypsin-EDTA (1X) (GIBCO, P/N 25300-054)</p>
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<p>Saponin (Sigma, P/N S4521)</p>
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<p>Staurosporine (Sigma, P/N S5921)</p>
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<p>Camptothecin (Sigma, P/N C9911)</p>
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<p><strong>III. Methods, Procedures, and Results</strong></p>
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<p>'See STEPS'</p>
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<p><strong>Result</strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hb5c2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hb5c2w6.png" data-ofn="Screen Shot 2016-12-16 at 11.27.01 AM.png" /></p>
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<div class="page" title="Page 3"><strong>References</strong></div>
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<p>Gani OA, Engh RA (2010) Protein kinase inhibition of clinically important staurosporine analogues. Nat Prod Rep. 27: 489-98</p>
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<p>Gescher A. (2000) Staurosporine analogues – pharmacological toys or useful antitumor agents? Crit Rev Oncol Hematol. 34: 127-35</p>
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<p>Venditto VJ, Simanek EE (2010) Cancer therapies utilizing the camptothecins: a review of the in vivo literature. Mol Pharm. 7: 307-49</p>
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[STEPS]
SECTION: Cell Preparation
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SECTION: Staurosporine Treatment
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SECTION: Staurosporine Treatment
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SECTION: Sapphire700 Staining
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SECTION: Sapphire700 Staining
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SECTION: Sapphire700 Staining
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SECTION: Staurosporine Treatment
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