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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="National Cancer Institutes Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]" /><meta name="citation_title" content="Hepatocyte Primary ROS Assay" /><meta name="citation_publisher" content="National Cancer Institute (US)" /><meta name="citation_date" content="2010/04" /><meta name="citation_author" content="Stephan T. Stern" /><meta name="citation_author" content="Banu S. Zolnik" /><meta name="citation_pmid" content="39013062" /><meta name="citation_doi" content="10.17917/0WF6-GX40" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK604946/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Hepatocyte Primary ROS Assay" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Cancer Institute (US)" /><meta name="DC.Contributor" content="Stephan T. Stern" /><meta name="DC.Contributor" content="Banu S. Zolnik" /><meta name="DC.Date" content="2010/04" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK604946/" /><meta name="description" content="This protocol describes the testing of nanoparticle formulations for reactive oxygen species (ROS) generation in male Sprague-Dawley (SD) primary hepatocytes, as part of the in vitro NCL preclinical characterization cascade. The protocol utilizes a fluorescent redox active probe. Primary hepatocytes were chosen since they have a greater metabolic activity than hepatocyte cell lines." /><meta name="og:title" content="Hepatocyte Primary ROS Assay" /><meta name="og:type" content="book" /><meta name="og:description" content="This protocol describes the testing of nanoparticle formulations for reactive oxygen species (ROS) generation in male Sprague-Dawley (SD) primary hepatocytes, as part of the in vitro NCL preclinical characterization cascade. The protocol utilizes a fluorescent redox active probe. Primary hepatocytes were chosen since they have a greater metabolic activity than hepatocyte cell lines." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK604946/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-nciprotocols-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/nciprotocols/ncipr13/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK604946/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/core/mathjax/2.7.9/MathJax.js?config=/corehtml/pmc/js/mathjax-config-classic.3.4.js"></script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>National Cancer Institutes Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. doi: 10.17917/0WF6-GX40</p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/nciprotocols/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-nciprotocols-lrg.png" alt="Cover of National Cancer Institutes Nanotechnology Characterization Laboratory Assay Cascade Protocols" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>National Cancer Institutes Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK604946_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK604946_dtls__"><div>Bethesda (MD): <a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&amp;targetsite=external&amp;targetcat=link&amp;targettype=publisher">National Cancer Institute (US)</a>; 2005 May 1-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/nciprotocols/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr15/" title="Previous page in this title">&lt; Prev</a><a class="active page_link next" href="/books/n/nciprotocols/ncipr12/" title="Next page in this title">Next &gt;</a></div></div></div></div></div>
<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK604946_"><span class="label">NCL Method GTA-7</span><span class="title" itemprop="name">Hepatocyte Primary ROS Assay</span></h1><div class="subtitle whole_rhythm">Version 1.1</div><p class="contrib-group"><span itemprop="author">Stephan T. Stern</span>, Ph.D. and <span itemprop="author">Banu S. Zolnik</span>, Ph.D.</p><p class="small">Published: <span itemprop="datePublished">April 2010</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="ncipr13.s1"><h2 id="_ncipr13_s1_">1. Introduction</h2><p>This protocol describes the testing of nanoparticle formulations for reactive oxygen species (ROS) generation in male Sprague-Dawley (SD) primary hepatocytes, as part of the <i>in vitro</i> NCL preclinical characterization cascade. The protocol utilizes a fluorescent redox active probe. Primary hepatocytes were chosen since they have a greater metabolic activity than hepatocyte cell lines.</p></div><div id="ncipr13.s2"><h2 id="_ncipr13_s2_">2. Principles</h2><p>Dichlorofluorescein diacetate (DCFH-DA) is a ROS probe that undergoes intracellular deacetylation, followed by ROS mediated oxidation to a fluorescent species (ex. 485 nm and em. 530 nm). DCFH-DA can be used to measure ROS generation in the cytoplasm and cellular organelles, such as the mitochondria. Fluorescence intensity is quantified in a microplate spectrophotometer (<a class="bk_pop" href="#ncipr13.ref1">1</a>).</p></div><div id="ncipr13.s3"><h2 id="_ncipr13_s3_">3. Reagents, Materials, Cell Lines, and Equipment</h2><blockquote><p><i>Note: The NCL does not endorse any of the suppliers listed below; their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted</i>.</p></blockquote><dl id="ncipr13.l1" class="temp-labeled-list"><dt>3.1.</dt><dd id="ncipr13.lt1"><p class="no_top_margin">Reagents
<dl id="ncipr13.l2" class="temp-labeled-list"><dt>3.1.1.</dt><dd id="ncipr13.lt2"><p class="no_top_margin">2&#x02019;,7&#x02019;-Dichlorodihydrofluoroscein Diacetate (DCFH-DA) (Molecular Probes, D399)</p></dd><dt>3.1.2.</dt><dd id="ncipr13.lt3"><p class="no_top_margin">Dimethyl sulfoxide (DMSO) (Aldrich, 154938)</p></dd><dt>3.1.3.</dt><dd id="ncipr13.lt4"><p class="no_top_margin">HyQ Phosphate Buffered Saline (PBS) (1X) (HyClone, SH30256.01)</p></dd><dt>3.1.4.</dt><dd id="ncipr13.lt5"><p class="no_top_margin">Diethyl maleate, 97% (DEM) (Aldrich, D97703-1006)</p></dd><dt>3.1.5.</dt><dd id="ncipr13.lt6"><p class="no_top_margin">Williams Media E (Sigma, W1878)</p></dd><dt>3.1.6.</dt><dd id="ncipr13.lt7"><p class="no_top_margin">L-glutamine (HyClone, SH30034.01)</p></dd><dt>3.1.7.</dt><dd id="ncipr13.lt8"><p class="no_top_margin">Penicillin/Streptomycin (Invitrogen, 15140-122)</p></dd><dt>3.1.8.</dt><dd id="ncipr13.lt9"><p class="no_top_margin">Fetal bovine serum (FBS) (HyClone, SH30070.03)</p></dd><dt>3.1.9.</dt><dd id="ncipr13.lt10"><p class="no_top_margin">Insulin (Sigma, I-1882)</p></dd><dt>3.1.10.</dt><dd id="ncipr13.lt11"><p class="no_top_margin">Dexamethasone (Sigma, D4902)</p></dd><dt>3.1.11.</dt><dd id="ncipr13.lt12"><p class="no_top_margin">ITS + Premix (insulin, human transferrin, and selenous acid) (BD Biosciences, 354352)</p></dd></dl></p></dd><dt>3.2.</dt><dd id="ncipr13.lt13"><p class="no_top_margin">Materials
<dl id="ncipr13.l3" class="temp-labeled-list"><dt>3.2.1.</dt><dd id="ncipr13.lt14"><p class="no_top_margin">Black Costar 96 well plates (Sigma, CLS3603)</p></dd></dl></p></dd><dt>3.3.</dt><dd id="ncipr13.lt15"><p class="no_top_margin">Cell Lines
<dl id="ncipr13.l4" class="temp-labeled-list"><dt>3.3.1.</dt><dd id="ncipr13.lt16"><p class="no_top_margin">Cryopreserved Male Sprague-Dawley primary heptocytes (Cellzdirect, RTCH-M).</p></dd></dl></p></dd><dt>3.4.</dt><dd id="ncipr13.lt17"><p class="no_top_margin">Equipment
<dl id="ncipr13.l5" class="temp-labeled-list"><dt>3.4.1.</dt><dd id="ncipr13.lt18"><p class="no_top_margin">Plate reader (Safire<sup>2</sup>&#x02013;Tecan or equivalent)</p></dd><dt>3.4.2.</dt><dd id="ncipr13.lt19"><p class="no_top_margin">Centrifuge set at 70 x g (Microfuge 22R Centrifuge-Beckman Coulter)</p></dd></dl></p></dd></dl></div><div id="ncipr13.s4"><h2 id="_ncipr13_s4_">4. Reagent and Control Preparation (Prepare immediately prior to use)</h2><dl id="ncipr13.l6" class="temp-labeled-list"><dt>4.1.</dt><dd id="ncipr13.lt20"><p class="no_top_margin">DEM Positive Control: prepare 5 mM DEM treatment solution in William&#x02019;s Medium E Maintenance Media (described in <a href="#ncipr13.lt27">Section 5.1.2</a>).</p></dd><dt>4.2.</dt><dd id="ncipr13.lt21"><p class="no_top_margin">ROS Fluorescent Probe reagent (Prepare in dark room, protect solutions from light!)
<dl id="ncipr13.l7" class="temp-labeled-list"><dt>4.2.1.</dt><dd id="ncipr13.lt22"><p class="no_top_margin">DCFH-DA Stock (10 mM): 5 mg in 1 mL of DMSO.</p></dd><dt>4.2.2.</dt><dd id="ncipr13.lt23"><p class="no_top_margin">DCFH-DA Working Stock (40 &#x000b5;M): <i>QS</i> 200 &#x000b5;L of 10 mM Stock to 50 mL in PBS buffer.</p></dd></dl></p></dd></dl></div><div id="ncipr13.s5"><h2 id="_ncipr13_s5_">5. Experimental Procedure</h2><dl id="ncipr13.l8" class="temp-labeled-list"><dt>5.1.</dt><dd id="ncipr13.lt24"><p class="no_top_margin">Prepare the two required media for the hepatocytes, as follows:
<dl id="ncipr13.l9" class="temp-labeled-list"><dt>5.1.1.</dt><dd id="ncipr13.lt25"><p class="no_top_margin">Thaw Media:
<dl id="ncipr13.l10" class="temp-labeled-list"><dt>5.1.1.1.</dt><dd id="ncipr13.lt26"><p class="no_top_margin">Add 100 &#x000b5;L of insulin stock (4 mg/mL) (stored at &#x02212;20&#x000b0;C) and 10 &#x000b5;L of 10 mM dexamethasone stock (stored at &#x02212;20&#x000b0;C) to 100 mL of William&#x02019;s Medium E with serum (2 mM L-glutamine, 50 U/mL penicillin, 50 &#x000b5;g/mL streptomycin and 5% FBS).</p></dd></dl></p></dd><dt>5.1.2.</dt><dd id="ncipr13.lt27"><p class="no_top_margin">Maintenance Media:
<dl id="ncipr13.l11" class="temp-labeled-list"><dt>5.1.2.1.</dt><dd id="ncipr13.lt28"><p class="no_top_margin">Add 1 mL of ITS+ (stored at +4&#x000b0;C) and 1 &#x000b5;L dexamethasone to 100 mL of William&#x02019;s Medium E (2 mM L-glutamine, 50 U/mL penicillin, and 50 &#x000b5;g/mL streptomycin)</p></dd></dl></p></dd></dl></p></dd><dt>5.2.</dt><dd id="ncipr13.lt29"><p class="no_top_margin">Cell Preparation:
<dl id="ncipr13.l12" class="temp-labeled-list"><dt>5.2.1.</dt><dd id="ncipr13.lt30"><p class="no_top_margin">Warm the Thaw Media to 37&#x000b0;C in the water bath and thaw the vial containing hepatocytes as follows:
<dl id="ncipr13.l13" class="temp-labeled-list"><dt>5.2.1.1.</dt><dd id="ncipr13.lt31"><p class="no_top_margin">Add a few milliliters of warm Thaw Media to a 50 mL conical tube, swirl the media, and aspirate off supernatant.</p></dd><dt>5.2.1.2.</dt><dd id="ncipr13.lt32"><p class="no_top_margin">Wipe the vial with 70% EtOH, loosen and retighten the cap.</p></dd><dt>5.2.1.3.</dt><dd id="ncipr13.lt33"><p class="no_top_margin">Swirl the vial containing cryopreserved cells in the water bath until only a small ice pellet remains (about 1 minute, 45 seconds).</p></dd><dt>5.2.1.4.</dt><dd id="ncipr13.lt34"><p class="no_top_margin">Wipe the vial with 70% EtOH and transfer the contents to the 50 mL conical tube.</p></dd><dt>5.2.1.5.</dt><dd id="ncipr13.lt35"><p class="no_top_margin">Add Thaw Media to the 50 mL conical tube as follows:
<ul id="ncipr13.l14"><li id="ncipr13.lt36" class="half_rhythm"><div>Add 1 mL by adding 200 &#x000b5;L at a time, swirling between additions.</div></li><li id="ncipr13.lt37" class="half_rhythm"><div>Add 5 mL by adding 500 &#x000b5;L at a time, swirling between additions.</div></li><li id="ncipr13.lt38" class="half_rhythm"><div>Add 5 mL by adding 1 mL at a time, swirling between additions.</div></li><li id="ncipr13.lt39" class="half_rhythm"><div><i>QS</i> the tube to 50 mL.</div></li><li id="ncipr13.lt40" class="half_rhythm"><div>Spin the cells at room temperature for 4 min at 70 x g.</div></li><li id="ncipr13.lt41" class="half_rhythm"><div>Carefully aspirate the supernatant and add 5 mL of Thaw media. Gently resuspend by pipetting.</div></li><li id="ncipr13.lt42" class="half_rhythm"><div>Count viable cell density using a hemocytometer.</div></li><li id="ncipr13.lt43" class="half_rhythm"><div>Dilute cells to a density of 7.5 &#x000d7; 10<sup>5</sup> cells/mL in Maintenance Media.</div></li><li id="ncipr13.lt44" class="half_rhythm"><div>Plate 150 &#x000b5;L cells/well as per plate format for time zero, 0.5, 1, 1.5, 2, 2.5 and 3 hour sample exposures (<a href="#ncipr13.app1">Appendix</a>).</div></li><li id="ncipr13.lt45" class="half_rhythm"><div>Incubate plates for 4 hours at 5% CO<sub>2</sub>, 37&#x000b0;C and 95% humidity (<a class="figpopup" href="/books/NBK604946/figure/ncipr13.fig1/?report=objectonly" target="object" rid-figpopup="figncipr13fig1" rid-ob="figobncipr13fig1">Figure 1</a>).</div></li></ul></p></dd></dl></p></dd></dl></p></dd></dl><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr13fig1" co-legend-rid="figlgndncipr13fig1"><a href="/books/NBK604946/figure/ncipr13.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr13fig1" rid-ob="figobncipr13fig1"><img class="small-thumb" src="/books/NBK604946/bin/ncipr13f1.gif" src-large="/books/NBK604946/bin/ncipr13f1.jpg" alt="Figure 1. Example of SD Primary Hepatocytes Cell Culture Appearance." /></a><div class="icnblk_cntnt" id="figlgndncipr13fig1"><h4 id="ncipr13.fig1"><a href="/books/NBK604946/figure/ncipr13.fig1/?report=objectonly" target="object" rid-ob="figobncipr13fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Example of SD Primary Hepatocytes Cell Culture Appearance. Image was taken with a phase contrast microscope at 250X magnification. </p></div></div></div><div id="ncipr13.s6"><h2 id="_ncipr13_s6_">6. Test Nanomaterial Addition</h2><dl id="ncipr13.l15" class="temp-labeled-list"><dt>6.1.</dt><dd id="ncipr13.lt46"><p class="no_top_margin">The highest concentration of nanomaterial tested should be at the limit of solubility. The test sample should be at physiological pH. Neutralization of acidic/basic test samples may be required.</p></dd><dt>6.2.</dt><dd id="ncipr13.lt47"><p class="no_top_margin">Dilute test compound in Maintenance Media, making a total of nine 1:4 dilutions.</p></dd><dt>6.3.</dt><dd id="ncipr13.lt48"><p class="no_top_margin"><b>(Work in the dark!)</b> Add 150 &#x000b5;L of 40 &#x000b5;M DCFH-DA to <strike>t</strike>est sample exposure plate containing 150 &#x000b5;L of Maintenance Media (Final concentration of DCFH-DA is 20 &#x000b5;M) and incubate cells for 30 min under standard culture conditions. Centrifuge the plates at 70 x g for 4 min without brake. Remove DCFH-DA and wash plate with 200 &#x000b5;L of Maintenance Media at 70 x g for 4 minutes with no deceleration. Read time zero measurement, then add 200 &#x000b5;L of each sample dilution to each plate as per plate format (<a href="#ncipr13.app1">Appendix</a>).</p></dd><dt>6.4.</dt><dd id="ncipr13.lt49"><p class="no_top_margin">ROS Assay Experimental Procedure <b>(do not expose plates to light!)</b>
<dl id="ncipr13.l16" class="temp-labeled-list"><dt>6.4.1.</dt><dd id="ncipr13.lt50"><p class="no_top_margin">Remove test plate at 0.5, 1, 2, and 3 h post exposure from the incubator and read at ex. 485 nm and em. 530 nm.</p></dd></dl></p></dd></dl></div><div id="ncipr13.s7"><h2 id="_ncipr13_s7_">7. Calculations</h2><dl id="ncipr13.l17" class="temp-labeled-list"><dt>7.1.</dt><dd id="ncipr13.lt51"><p class="no_top_margin">Rows D and E are used as cell-free blanks, which are subtracted from the corresponding sample and control columns (e.g., A1-D1 or B2-D2; see <a href="#ncipr13.app1">Appendix</a>).</p></dd><dt>7.2.</dt><dd id="ncipr13.lt52"><p class="no_top_margin">Wells 1(A-C) and 12(A-C) are the media controls, and wells 11(A-C) are the DEM positive controls for samples in wells 2(A-C) - 10(A-C). Wells 1(F-H) and 12(F-H) are the media controls, and wells 11(F-H) are the positive controls for samples in wells 2(F-H) - 10(F-H) (see <a href="#ncipr13.app1">Appendix</a>).
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr13.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr13.eq1" display="block"><mrow><mi>%</mi><mtext>&#x02009;</mtext><mi>C</mi><mi>o</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>o</mi><mi>l</mi><mtext>&#x02009;</mtext><mi>f</mi><mi>l</mi><mi>u</mi><mi>o</mi><mi>r</mi><mi>e</mi><mi>s</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>c</mi><mi>e</mi><mo>=</mo><mfrac><mrow><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext>&#x02009;</mtext><mi>f</mi><mi>l</mi><mi>u</mi><mi>o</mi><mi>r</mi><mi>e</mi><mi>s</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>c</mi><mi>e</mi></mrow><mrow><mi>m</mi><mi>e</mi><mi>d</mi><mi>i</mi><mi>a</mi><mtext>&#x02009;</mtext><mi>c</mi><mi>o</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>o</mi><mi>l</mi><mtext>&#x02009;</mtext><mi>f</mi><mi>l</mi><mi>u</mi><mi>o</mi><mi>r</mi><mi>e</mi><mi>s</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>c</mi><mi>e</mi></mrow></mfrac><mo>*</mo><mn>100</mn></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div>
Mean, SD and %CV should be calculated for each positive control and unknown sample.</p></dd></dl></div><div id="ncipr13.s8"><h2 id="_ncipr13_s8_">8. Acceptance Criteria</h2><dl id="ncipr13.l18" class="temp-labeled-list"><dt>8.1.</dt><dd id="ncipr13.lt53"><p class="no_top_margin">DCFH-DA fluorescence for the DEM positive control should be at least 140 % of media control at 2 hours.</p></dd><dt>8.2.</dt><dd id="ncipr13.lt54"><p class="no_top_margin">The positive control and sample replicate coefficient of variations should be within 50%.</p></dd><dt>8.3.</dt><dd id="ncipr13.lt55"><p class="no_top_margin">The assay is acceptable if condition <a href="#ncipr13.lt53">8.1</a> and <a href="#ncipr13.lt54">8.2</a> are met. Otherwise, the assay should be repeated until acceptance criteria are met.</p></dd></dl></div><div id="ncipr13.rl.r1"><h2 id="_ncipr13_rl_r1_">9. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ncipr13.ref1">Black, M.J. and Brandt, R.B., Spectrofluorometric analysis of hydrogen peroxide, <em>Anal. Biochem.</em>, 58, 246, 1974.
[<a href="https://pubmed.ncbi.nlm.nih.gov/4825377" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 4825377</span></a>]</div></dd></dl></div><div id="ncipr13.s9"><h2 id="_ncipr13_s9_">10. Abbreviations</h2><dl><dt id="ncipr13.abb_DL1_DI1">APAP</dt><dd><p>acetaminophen</p></dd><dt id="ncipr13.abb_DL1_DI2">CV</dt><dd><p>coefficient of variation</p></dd><dt id="ncipr13.abb_DL1_DI3">DCFH-DA</dt><dd><p>dichlorofluorescein diacetate</p></dd><dt id="ncipr13.abb_DL1_DI4">DEM</dt><dd><p>diethyl maleate</p></dd><dt id="ncipr13.abb_DL1_DI5">DMSO</dt><dd><p>dimethyl sulfoxide</p></dd><dt id="ncipr13.abb_DL1_DI6">FBS</dt><dd><p>fetal bovine serum</p></dd><dt id="ncipr13.abb_DL1_DI7">INT</dt><dd><p>2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride</p></dd><dt id="ncipr13.abb_DL1_DI8">LDH</dt><dd><p>lactate dehydrogenase</p></dd><dt id="ncipr13.abb_DL1_DI9">LLC-PK1 cells</dt><dd><p>renal epithelial cell line, porcine kidney</p></dd><dt id="ncipr13.abb_DL1_DI10">MTT</dt><dd><p>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</p></dd><dt id="ncipr13.abb_DL1_DI11">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr13.abb_DL1_DI12">ROS</dt><dd><p>reactive oxygen species</p></dd><dt id="ncipr13.abb_DL1_DI13">SD</dt><dd><p>standard deviation</p></dd></dl></div><div id="ncipr13.app1"><h2 id="_ncipr13_app1_">11. Appendix</h2><p>Example of a 96-well plate template.</p><div id="ncipr13.app1.fig1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ncipr13app1f1&amp;p=BOOKS&amp;id=604946_ncipr13app1f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK604946/bin/ncipr13app1f1.jpg" alt="Image ncipr13app1f1" class="tileshop" title="Click on image to zoom" /></a></div></div></div><div><dl class="temp-labeled-list small"><dt></dt><dd><div><p class="no_top_margin"><p>This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.</p></p></div></dd><dt></dt><dd><div><p class="no_top_margin"><div>
<span class="mixed-citation" id="ncipr13.suggestedcitation">Stern ST, Zolnik BS, NCL Method GTA-7: Hepatocyte Primary ROS Assay. <a href="https://ncl.cancer.gov/resources/assay-cascade-protocols" ref="pagearea=body&amp;targetsite=external&amp;targetcat=link&amp;targettype=uri">https://ncl.cancer.gov/resources/assay-cascade-protocols</a> DOI: <a href="http://dx.crossref.org/10.17917/0WF6-GX40" ref="pagearea=body&amp;targetsite=external&amp;targetcat=link&amp;targettype=uri">10.17917/0WF6-GX40</a></span>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK604946</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/39013062" title="PubMed record of this page" ref="pagearea=meta&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">39013062</a></span>DOI: <a href="http://dx.crossref.org/10.17917/0WF6-GX40" ref="pagearea=body&amp;targetsite=external&amp;targetcat=link&amp;targettype=uri">10.17917/0WF6-GX40</a></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr15/" title="Previous page in this title">&lt; Prev</a><a class="active page_link next" href="/books/n/nciprotocols/ncipr12/" title="Next page in this title">Next &gt;</a></div></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK604946/?report=reader">PubReader</a></li><li><a href="/books/NBK604946/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK604946" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK604946" style="display:none" title="Cite this Page"><div class="bk_tt">Stern ST, Zolnik BS. Hepatocyte Primary ROS Assay: Version 1.1. 2010 Apr. In: National Cancer Institutes Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method GTA-7.<span class="bk_cite_avail"></span> doi: 10.17917/0WF6-GX40</div></div></li><li><a href="/books/NBK604946/pdf/Bookshelf_NBK604946.pdf">PDF version of this page</a> (741K)</li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ncipr13.s1" ref="log$=inpage&amp;link_id=inpage">Introduction</a></li><li><a href="#ncipr13.s2" ref="log$=inpage&amp;link_id=inpage">Principles</a></li><li><a href="#ncipr13.s3" ref="log$=inpage&amp;link_id=inpage">Reagents, Materials, Cell Lines, and Equipment</a></li><li><a href="#ncipr13.s4" ref="log$=inpage&amp;link_id=inpage">Reagent and Control Preparation (Prepare immediately prior to use)</a></li><li><a href="#ncipr13.s5" ref="log$=inpage&amp;link_id=inpage">Experimental Procedure</a></li><li><a href="#ncipr13.s6" ref="log$=inpage&amp;link_id=inpage">Test Nanomaterial Addition</a></li><li><a href="#ncipr13.s7" ref="log$=inpage&amp;link_id=inpage">Calculations</a></li><li><a href="#ncipr13.s8" ref="log$=inpage&amp;link_id=inpage">Acceptance Criteria</a></li><li><a href="#ncipr13.rl.r1" ref="log$=inpage&amp;link_id=inpage">References</a></li><li><a href="#ncipr13.s9" ref="log$=inpage&amp;link_id=inpage">Abbreviations</a></li><li><a href="#ncipr13.app1" ref="log$=inpage&amp;link_id=inpage">Appendix</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div 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