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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Hepatocyte Primary ROS Assay - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]">
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<meta name="citation_title" content="Hepatocyte Primary ROS Assay">
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<meta name="citation_publisher" content="National Cancer Institute (US)">
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<meta name="citation_date" content="2010/04">
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<meta name="citation_author" content="Stephan T. Stern">
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<meta name="citation_author" content="Banu S. Zolnik">
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<meta name="citation_pmid" content="39013062">
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<meta name="citation_doi" content="10.17917/0WF6-GX40">
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<meta name="DC.Title" content="Hepatocyte Primary ROS Assay">
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<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.">
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<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.">
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id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="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="contribs">Stern ST, Zolnik BS.</p><p class="fm-aai"><a href="#_NBK604946_pubdet_">Publication Details</a></p></div></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="bibr" href="#ncipr13.ref1" rid="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"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr13.lt1"><p class="no_top_margin">Reagents
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<dl id="ncipr13.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.1.</dt><dd id="ncipr13.lt2"><p class="no_top_margin">2’,7’-Dichlorodihydrofluoroscein Diacetate (DCFH-DA) (Molecular Probes, D399)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.2.</dt><dd id="ncipr13.lt3"><p class="no_top_margin">Dimethyl sulfoxide (DMSO) (Aldrich, 154938)</p></dd></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><dt>3.1.4.</dt><dd id="ncipr13.lt5"><p class="no_top_margin">Diethyl maleate, 97% (DEM) (Aldrich, D97703-1006)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.5.</dt><dd id="ncipr13.lt6"><p class="no_top_margin">Williams Media E (Sigma, W1878)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.6.</dt><dd id="ncipr13.lt7"><p class="no_top_margin">L-glutamine (HyClone, SH30034.01)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.7.</dt><dd id="ncipr13.lt8"><p class="no_top_margin">Penicillin/Streptomycin (Invitrogen, 15140-122)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.8.</dt><dd id="ncipr13.lt9"><p class="no_top_margin">Fetal bovine serum (FBS) (HyClone, SH30070.03)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.9.</dt><dd id="ncipr13.lt10"><p class="no_top_margin">Insulin (Sigma, I-1882)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.10.</dt><dd id="ncipr13.lt11"><p class="no_top_margin">Dexamethasone (Sigma, D4902)</p></dd></dl><dl class="bkr_refwrap"><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></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr13.lt13"><p class="no_top_margin">Materials
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<dl id="ncipr13.l3" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.2.1.</dt><dd id="ncipr13.lt14"><p class="no_top_margin">Black Costar 96 well plates (Sigma, CLS3603)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr13.lt15"><p class="no_top_margin">Cell Lines
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<dl id="ncipr13.l4" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.4.</dt><dd id="ncipr13.lt17"><p class="no_top_margin">Equipment
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<dl id="ncipr13.l5" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.4.1.</dt><dd id="ncipr13.lt18"><p class="no_top_margin">Plate reader (Safire<sup>2</sup>–Tecan or equivalent)</p></dd></dl><dl class="bkr_refwrap"><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></dl></p></dd></dl></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"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr13.lt20"><p class="no_top_margin">DEM Positive Control: prepare 5 mM DEM treatment solution in William’s Medium E Maintenance Media (described in <a href="#ncipr13.lt27">Section 5.1.2</a>).</p></dd></dl><dl class="bkr_refwrap"><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!)
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<dl id="ncipr13.l7" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><dt>4.2.2.</dt><dd id="ncipr13.lt23"><p class="no_top_margin">DCFH-DA Working Stock (40 µM): <i>QS</i> 200 µL of 10 mM Stock to 50 mL in PBS buffer.</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr13.s5"><h2 id="_ncipr13_s5_">5. Experimental Procedure</h2><dl id="ncipr13.l8" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.</dt><dd id="ncipr13.lt24"><p class="no_top_margin">Prepare the two required media for the hepatocytes, as follows:
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<dl id="ncipr13.l9" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.1.</dt><dd id="ncipr13.lt25"><p class="no_top_margin">Thaw Media:
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<dl id="ncipr13.l10" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.1.1.</dt><dd id="ncipr13.lt26"><p class="no_top_margin">Add 100 µL of insulin stock (4 mg/mL) (stored at −20°C) and 10 µL of 10 mM dexamethasone stock (stored at −20°C) to 100 mL of William’s Medium E with serum (2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin and 5% FBS).</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>5.1.2.</dt><dd id="ncipr13.lt27"><p class="no_top_margin">Maintenance Media:
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<dl id="ncipr13.l11" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.2.1.</dt><dd id="ncipr13.lt28"><p class="no_top_margin">Add 1 mL of ITS+ (stored at +4°C) and 1 µL dexamethasone to 100 mL of William’s Medium E (2 mM L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin)</p></dd></dl></dl></p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>5.2.</dt><dd id="ncipr13.lt29"><p class="no_top_margin">Cell Preparation:
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<dl id="ncipr13.l12" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.2.1.</dt><dd id="ncipr13.lt30"><p class="no_top_margin">Warm the Thaw Media to 37°C in the water bath and thaw the vial containing hepatocytes as follows:
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<dl id="ncipr13.l13" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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:
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<ul id="ncipr13.l14"><li id="ncipr13.lt36" class="half_rhythm"><div>Add 1 mL by adding 200 µL at a time, swirling between additions.</div></li><li id="ncipr13.lt37" class="half_rhythm"><div>Add 5 mL by adding 500 µL 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 × 10<sup>5</sup> cells/mL in Maintenance Media.</div></li><li id="ncipr13.lt44" class="half_rhythm"><div>Plate 150 µL 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°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></dl></p></dd></dl></dl></p></dd></dl></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"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><dt>6.3.</dt><dd id="ncipr13.lt48"><p class="no_top_margin"><b>(Work in the dark!)</b> Add 150 µL of 40 µM DCFH-DA to <strike>t</strike>est sample exposure plate containing 150 µL of Maintenance Media (Final concentration of DCFH-DA is 20 µM) 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 µL of Maintenance Media at 70 x g for 4 minutes with no deceleration. Read time zero measurement, then add 200 µL of each sample dilution to each plate as per plate format (<a href="#ncipr13.app1">Appendix</a>).</p></dd></dl><dl class="bkr_refwrap"><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>
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<dl id="ncipr13.l16" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl></p></dd></dl></dl></div><div id="ncipr13.s7"><h2 id="_ncipr13_s7_">7. Calculations</h2><dl id="ncipr13.l17" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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>).
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<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> </mtext><mi>C</mi><mi>o</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>o</mi><mi>l</mi><mtext> </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> </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> </mtext><mi>c</mi><mi>o</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>o</mi><mi>l</mi><mtext> </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>
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Mean, SD and %CV should be calculated for each positive control and unknown sample.</p></dd></dl></dl></div><div id="ncipr13.s8"><h2 id="_ncipr13_s8_">8. Acceptance Criteria</h2><dl id="ncipr13.l18" class="temp-labeled-list"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><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></dl></div><div id="ncipr13.rl.r1"><h2 id="_ncipr13_rl_r1_">9. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><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.
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[<a href="https://pubmed.ncbi.nlm.nih.gov/4825377" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 4825377</span></a>]</div></dd></dl></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&p=BOOKS&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"><dl class="bkr_refwrap"><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></dl><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_top_margin"><div>
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<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&targetsite=external&targetcat=link&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&targetsite=external&targetcat=link&targettype=uri">10.17917/0WF6-GX40</a></span>
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</div></p></div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK604946_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Stephan T. Stern</span>, Ph.D. and <span itemprop="author">Banu S. Zolnik</span>, Ph.D.</p><h3>Publication History</h3><p class="small">Published: <span itemprop="datePublished">April 2010</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p><a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Cancer Institute (US)</a>, Bethesda (MD)</p><h3>NLM Citation</h3><p>Stern ST, Zolnik BS. Hepatocyte Primary ROS Assay: Version 1.1. 2010 Apr. In: National Cancer Institute’s 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</p></div><div class="small-screen-prev"><a href="/books/n/nciprotocols/ncipr15/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/nciprotocols/ncipr12/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobncipr13fig1"><div id="ncipr13.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Example%20of%20SD%20Primary%20Hepatocytes%20Cell%20Culture%20Appearance.&p=BOOKS&id=604946_ncipr13f1.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 data-src="/books/NBK604946/bin/ncipr13f1.jpg" alt="Figure 1. Example of SD Primary Hepatocytes Cell Culture Appearance." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Example of SD Primary Hepatocytes Cell Culture Appearance</span></h3><div class="caption"><p>Image was taken with a phase contrast microscope at 250X magnification.</p></div></div></article><article data-type="fig" id="figobncipr13app1fig1"><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&p=BOOKS&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 data-src="/books/NBK604946/bin/ncipr13app1f1.jpg" alt="Image ncipr13app1f1" class="tileshop" title="Click on image to zoom" /></a></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script><script type="text/javascript" src="/core/mathjax/2.7.9/MathJax.js?config=TeX-AMS-MML_SVG"> </script></div></div>
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