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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>LLC-PK1 Kidney Cell Apoptosis 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="LLC-PK1 Kidney Cell Apoptosis 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="Timothy M. Potter">
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<meta name="citation_pmid" content="39012989">
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<meta name="citation_doi" content="10.17917/E9YS-N327">
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<meta name="DC.Contributor" content="Timothy M. Potter">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button 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="_NBK604911_"><span class="label">NCL Method GTA-5</span><span class="title" itemprop="name">LLC-PK1 Kidney Cell Apoptosis Assay</span></h1><div class="subtitle whole_rhythm">Version 1.1</div><p class="contribs">Stern ST, Potter TM.</p><p class="fm-aai"><a href="#_NBK604911_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="ncipr11.s1"><h2 id="_ncipr11_s1_">1. Introduction</h2><p>This protocol describes the monitoring of nanoparticle treated porcine renal proximal tubule cells (LLC-PK1) for apoptosis, as part of the <i>in vitro</i> NCL preclinical characterization cascade. The protocol utilizes a fluorescent method to determine the degree of caspase-3 activation.</p></div><div id="ncipr11.s2"><h2 id="_ncipr11_s2_">2. Principles</h2><p>Caspase-3 Fluorometric Protease Assay:</p><p>Apoptosis in mammalian cells is initiated by activation of the caspase family of cysteine proteases. This assay quantifies caspase-3 activation <i>in vitro</i> by measuring the cleavage of caspase-3 substrate DEVD-7-amino-4-trifluoromethyl coumarin (AFC) to free AFC, which emits yellow-green fluorescence (λ<sub>max</sub> = 505 nm). This free AFC is measured using a microtiter plate reader (<a class="bibr" href="#ncipr11.ref1" rid="ncipr11.ref1">1</a>–<a class="bibr" href="#ncipr11.ref3" rid="ncipr11.ref3">3</a>).</p></div><div id="ncipr11.s3"><h2 id="_ncipr11_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="ncipr11.l1" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr11.lt1"><p class="no_top_margin">Reagents
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<dl id="ncipr11.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.1.</dt><dd id="ncipr11.lt2"><p class="no_top_margin">Cisplatin (Sigma, P4394)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.2.</dt><dd id="ncipr11.lt3"><p class="no_top_margin">M199 Cell Culture Media (Cambrex Cat. 12-109-F)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.3.</dt><dd id="ncipr11.lt4"><p class="no_top_margin">Quick Start Bradford Dye Reagent, 1X (Bio-Rad Lab., Inc., Cat. #500-0205)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.4.</dt><dd id="ncipr11.lt5"><p class="no_top_margin">Biovision Caspase-3 Fluorometeric Assay Kit (Biovision Cat. # K105-25)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.5.</dt><dd id="ncipr11.lt6"><p class="no_top_margin">Nanoparticle</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.6.</dt><dd id="ncipr11.lt7"><p class="no_top_margin">Fetal Bovine Serum (FBS) (Hyclone, SH30070.03)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.7.</dt><dd id="ncipr11.lt8"><p class="no_top_margin">Dimethyl sulfoxide (DMSO) (Aldrich# 154938)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr11.lt9"><p class="no_top_margin">Materials
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<dl id="ncipr11.l3" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.2.1.</dt><dd id="ncipr11.lt10"><p class="no_top_margin">Costar 6 well flat bottom cell culture plates (Costar, 3506)</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.2.</dt><dd id="ncipr11.lt11"><p class="no_top_margin">Costar 96 well flat bottom cell culture plates (Costar, 3598)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr11.lt12"><p class="no_top_margin">Cell Lines
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<dl id="ncipr11.l4" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.3.1.</dt><dd id="ncipr11.lt13"><p class="no_top_margin">LLC-PK1 (pig kidney cells) (ATCC, CL-101)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.4.</dt><dd id="ncipr11.lt14"><p class="no_top_margin">Equipment
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<dl id="ncipr11.l5" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.4.1.</dt><dd id="ncipr11.lt15"><p class="no_top_margin">Fluorescent Plate reader (Safire<sup>2</sup>–Tecan or equivalent)</p></dd></dl><dl class="bkr_refwrap"><dt>3.4.2.</dt><dd id="ncipr11.lt16"><p class="no_top_margin">Centrifuge set at 700-800 x g (Allegra X-15R- Beckman Coulter)</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr11.s4"><h2 id="_ncipr11_s4_">4. Reagent and Control Preparation</h2><dl id="ncipr11.l6" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr11.lt17"><p class="no_top_margin">Positive control
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<dl id="ncipr11.l7" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.1.</dt><dd id="ncipr11.lt18"><p class="no_top_margin">50 µM Cisplatin positive control: Prepare 50 mM cisplatin in DMSO. Dilute 50 mM stock to 50 µM in M199 media containing 3% FBS. Sterile filter using a 0.2 µm filter.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>4.2.</dt><dd id="ncipr11.lt19"><p class="no_top_margin">Solutions to make up prior (from the Assay kit, <a href="#ncipr11.lt6">Step 3.1.5</a>) (can be stored at 4°C for 6 months)
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<dl id="ncipr11.l8" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.2.1.</dt><dd id="ncipr11.lt20"><p class="no_top_margin">Add 10 µL of the DTT solution to 1 mL of the 2X Reaction Buffer.</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.2.</dt><dd id="ncipr11.lt21"><p class="no_top_margin">Thaw the Cell Lysis buffer and store at 4°C.</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.3.</dt><dd id="ncipr11.lt22"><p class="no_top_margin">DEVD-AFC is light sensitive, store protected from light.</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr11.s5"><h2 id="_ncipr11_s5_">5. Experimental Procedure</h2><dl id="ncipr11.l9" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.</dt><dd id="ncipr11.lt23"><p class="no_top_margin">Cell Preparation (or as recommended by supplier)
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<dl id="ncipr11.l10" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.1.</dt><dd id="ncipr11.lt24"><p class="no_top_margin">Harvest cryopreserved cells from prepared flasks <b>(limit to 20 passages)</b> (<a class="figpopup" href="/books/NBK604911/figure/ncipr11.fig1/?report=objectonly" target="object" rid-figpopup="figncipr11fig1" rid-ob="figobncipr11fig1">Figure 1</a>).</p></dd></dl><dl class="bkr_refwrap"><dt>5.1.2.</dt><dd id="ncipr11.lt25"><p class="no_top_margin">Count cell concentration using a coulter counter or hemocytometer.</p></dd></dl><dl class="bkr_refwrap"><dt>5.1.3.</dt><dd id="ncipr11.lt26"><p class="no_top_margin">Dilute cells to a density of 2.5 × 10<sup>5</sup> cells/mL in M199 (3% FBS) cell culture media.</p></dd></dl><dl class="bkr_refwrap"><dt>5.1.4.</dt><dd id="ncipr11.lt27"><p class="no_top_margin">Plate 2 mL of diluted cells to each well of a 6-well plate (5 × 10<sup>5</sup> cells/well). Test samples, positive controls, and media controls are run in triplicate. Timepoints are 0 h (media control), 6 h (sample and media control), 24 h (sample, positive control, and media control), and 48 h (sample and media control) for a total of 24 wells (see <a href="#ncipr11.appa">Appendix A</a>).</p></dd></dl><dl class="bkr_refwrap"><dt>5.1.5.</dt><dd id="ncipr11.lt28"><p class="no_top_margin">Incubate plates for 24 h at 5% CO<sub>2</sub>, 37°C and 95% humidity (<b>cells should be approximately 80% confluent</b>).</p></dd></dl><dl class="bkr_refwrap"><dt>5.1.6.</dt><dd id="ncipr11.lt29"><p class="no_top_margin">Replace cell culture media with 2 mL media containing blank media, test material or positive control. Desired test sample concentration is determined from LLC-PK1 Kidney Cytotoxicity Assay (NCL Method GTA-1). Treat cells for designated time period.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>5.2.</dt><dd id="ncipr11.lt30"><p class="no_top_margin">Caspase Activation Assay
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<dl id="ncipr11.l11" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.2.1.</dt><dd id="ncipr11.lt31"><p class="no_top_margin">Wash well with 1 mL of room temperature PBS.</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.2.</dt><dd id="ncipr11.lt32"><p class="no_top_margin">Add 200 µL ice cold lysis buffer to the well, scrape cells and collect in 0.6 mL eppendorf tubes.</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.3.</dt><dd id="ncipr11.lt33"><p class="no_top_margin">Incubate on ice for 10 minutes and centrifuge at 2000 x g for 5 minutes</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.4.</dt><dd id="ncipr11.lt34"><p class="no_top_margin">Transfer 50 µL of supernatant to a 96 well plate reader using the plating format described in <a href="#ncipr11.appb">Appendix B</a>. Add 50 µL of 2X Reaction Buffer (with DTT) to each sample well. Retain remaining cell lysate for protein determination.</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.5.</dt><dd id="ncipr11.lt35"><p class="no_top_margin">Add 5 µL of DEVD-AFC substrate (50 µM final concentration) and incubate at 37°C for 1-2 hours.</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.6.</dt><dd id="ncipr11.lt36"><p class="no_top_margin">Read at fluorescence at ex. 415 nm and em. 505 nm on a microtiter plate reader.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>5.3.</dt><dd id="ncipr11.lt37"><p class="no_top_margin">Protein Determination (Bradford Assay)
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<dl id="ncipr11.l12" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.3.1.</dt><dd id="ncipr11.lt38"><p class="no_top_margin">Dilute the 2 mg/mL BSA standard to make a standard curve from 0.125-1.0 mg/mL in 0.5 N NaOH.</p></dd></dl><dl class="bkr_refwrap"><dt>5.3.2.</dt><dd id="ncipr11.lt39"><p class="no_top_margin">Add 5 µL of standard (from <a href="#ncipr11.lt38">step 5.3.1</a>), cell lysate (from <a href="#ncipr11.lt33">step 5.2.3</a>), or water blank to each well of a microtiter plate in duplicate according the template in <a href="#ncipr11.appc">Appendix C</a>.</p></dd></dl><dl class="bkr_refwrap"><dt>5.3.3.</dt><dd id="ncipr11.lt40"><p class="no_top_margin">Add 250 µL of 1X Dye Reagent to each well of the plate.</p></dd></dl><dl class="bkr_refwrap"><dt>5.3.4.</dt><dd id="ncipr11.lt41"><p class="no_top_margin">Incubate at room temperature for at least 5 min and not longer than 1 h.</p></dd></dl><dl class="bkr_refwrap"><dt>5.3.5.</dt><dd id="ncipr11.lt42"><p class="no_top_margin">Read absorbance on a microtiter plate reader at 595 nm.</p></dd></dl></dl></p></dd></dl></dl><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr11fig1" co-legend-rid="figlgndncipr11fig1"><a href="/books/NBK604911/figure/ncipr11.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr11fig1" rid-ob="figobncipr11fig1"><img class="small-thumb" src="/books/NBK604911/bin/ncipr11f1.gif" src-large="/books/NBK604911/bin/ncipr11f1.jpg" alt="Figure 1. Example of LLC-PK1 Cell Culture Appearance." /></a><div class="icnblk_cntnt" id="figlgndncipr11fig1"><h4 id="ncipr11.fig1"><a href="/books/NBK604911/figure/ncipr11.fig1/?report=objectonly" target="object" rid-ob="figobncipr11fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Example of LLC-PK1 Cell Culture Appearance. Image was taken with a phase contrast microscope at 225X magnification. LLC-PK1 cells are approximately 80% confluent at this stage. </p></div></div></div><div id="ncipr11.s6"><h2 id="_ncipr11_s6_">6. Calculations</h2><dl id="ncipr11.l13" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>6.1.</dt><dd id="ncipr11.lt43"><p class="no_top_margin">Protein concentration is determined from the BSA standard curve following linear regression analysis (y = x(slope) + y int). Total protein is determined from the equation:
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr11.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr11.eq1" display="block"><mrow><mi>T</mi><mi>o</mi><mi>t</mi><mi>a</mi><mi>l</mi><mtext> </mtext><mi>l</mi><mi>y</mi><mi>s</mi><mi>a</mi><mi>t</mi><mi>e</mi><mtext> </mtext><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi><mtext> </mtext><mrow><mo>(</mo><mrow><mi>m</mi><mi>g</mi></mrow><mo>)</mo></mrow><mo>=</mo><mrow><mo>(</mo><mrow><mfrac><mrow><mi>m</mi><mi>g</mi><mtext> </mtext><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow><mrow><mi>m</mi><mi>L</mi></mrow></mfrac></mrow><mo>)</mo></mrow><mo>*</mo><mn>0.05</mn><mi>m</mi><mi>L</mi><mtext> </mtext><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>v</mi><mi>o</mi><mi>l</mi><mi>u</mi><mi>m</mi><mi>e</mi></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></p></dd></dl><dl class="bkr_refwrap"><dt>6.2.</dt><dd id="ncipr11.lt44"><p class="no_top_margin">Total Protein Normalized Caspase Activity:
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr11.deq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr11.eq2" 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>A</mi><mi>c</mi><mi>t</mi><mi>i</mi><mi>v</mi><mi>i</mi><mi>t</mi><mi>y</mi><mo>=</mo><mo stretchy="false">(</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>t</mi><mi>o</mi><mi>t</mi><mi>a</mi><mi>l</mi><mtext> </mtext><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>l</mi><mi>y</mi><mi>s</mi><mi>a</mi><mi>t</mi><mi>e</mi><mtext> </mtext><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow></mfrac><mo stretchy="false">)</mo><mo>/</mo><mo stretchy="false">(</mo><mfrac><mrow><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>o</mi><mi>f</mi><mtext> </mtext><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><mrow><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>o</mi><mi>f</mi><mtext> </mtext><mi>t</mi><mi>o</mi><mi>t</mi><mi>a</mi><mi>l</mi><mtext> </mtext><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>l</mi><mi>y</mi><mi>s</mi><mi>a</mi><mi>t</mi><mi>e</mi><mtext> </mtext><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow></mfrac><mo stretchy="false">)</mo><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 sample.</p></dd></dl></dl></div><div id="ncipr11.s7"><h2 id="_ncipr11_s7_">7. Acceptance Criteria</h2><dl id="ncipr11.l14" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>7.1.</dt><dd id="ncipr11.lt45"><p class="no_top_margin">The fold change in caspase activity at 24 hours for the cisplatin positive control versus media negative control should be at least 10.</p></dd></dl><dl class="bkr_refwrap"><dt>7.2.</dt><dd id="ncipr11.lt46"><p class="no_top_margin">The positive, media control, and sample replicate coefficient of variations should be within 50%.</p></dd></dl><dl class="bkr_refwrap"><dt>7.3.</dt><dd id="ncipr11.lt47"><p class="no_top_margin">The assay is acceptable if condition <a href="#ncipr11.lt45">7.1</a> and <a href="#ncipr11.lt46">7.2</a> are met. Otherwise, the assay should be repeated until acceptance criteria are met.</p></dd></dl><dl class="bkr_refwrap"><dt>7.4.</dt><dd id="ncipr11.lt48"><p class="no_top_margin">If statistical analysis determines that the total protein normalized control and treated fluorescence are significantly different from one another, then the fold change in fluorescence can be considered meaningful. This result would indicate that sample treatment significantly affected cell apoptosis.</p></dd></dl></dl></div><div id="ncipr11.rl.r1"><h2 id="_ncipr11_rl_r1_">8. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ncipr11.ref1">ISO 10993-5 Biological evaluation of medical devices: Part 5 Tests for <em>in vitro</em> cytotoxicity.</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ncipr11.ref2">F1903 – 98 Standard Practice for Testing for Biological Responses to Particles <em>in vitro</em>.</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ncipr11.ref3">Wang
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et al. (2005) Cell Bio. Int.
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29: 489–496.
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[<a href="https://pubmed.ncbi.nlm.nih.gov/15939633" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15939633</span></a>]</div></dd></dl></dl></div><div id="ncipr11.s8"><h2 id="_ncipr11_s8_">9. Abbreviations</h2><dl><dt id="ncipr11.abb_DL1_DI1">AFC</dt><dd><p>7-amino-trifluoromethyl coumarin</p></dd><dt id="ncipr11.abb_DL1_DI2">BSA</dt><dd><p>bovine serum albumin</p></dd><dt id="ncipr11.abb_DL1_DI3">CV</dt><dd><p>coefficient of variation</p></dd><dt id="ncipr11.abb_DL1_DI4">DEVD</dt><dd><p>aspartic acid-glutamic acid-valine-aspartic acid</p></dd><dt id="ncipr11.abb_DL1_DI5">DMSO</dt><dd><p>dimethyl sulfoxide</p></dd><dt id="ncipr11.abb_DL1_DI6">DTT</dt><dd><p>dithiothreitol</p></dd><dt id="ncipr11.abb_DL1_DI7">em.</dt><dd><p>Emission</p></dd><dt id="ncipr11.abb_DL1_DI8">ex.</dt><dd><p>Excitation</p></dd><dt id="ncipr11.abb_DL1_DI9">FBS</dt><dd><p>fetal bovine serum</p></dd><dt id="ncipr11.abb_DL1_DI10">LLC-PK1</dt><dd><p>porcine kidney cells</p></dd><dt id="ncipr11.abb_DL1_DI11">λ<sub>max</sub></dt><dd><p>maximal wavelength</p></dd><dt id="ncipr11.abb_DL1_DI12">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr11.abb_DL1_DI13">SD</dt><dd><p>standard deviation</p></dd></dl></div><div id="ncipr11.appendixesappgroup1"><h2 id="_ncipr11_appendixesappgroup1_">10. Appendices</h2><div id="ncipr11.appa"><h3>Appendix A</h3><p>Example of 6-well plate templates.</p><div id="ncipr11.appa.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=All%20samples%20are%20run%20in%20triplicate&p=BOOKS&id=604911_ncipr11appaf1.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/NBK604911/bin/ncipr11appaf1.jpg" alt="All samples are run in triplicate" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>All samples are run in triplicate. The following timepoints are recommended: 0 hr (media control), 6 hr (sample and media control), 24 hr (sample, positive control, and media control), and 48 hr (sample and media control) in which case 5 plates will be necessary.</p></div></div></div><div id="ncipr11.appb"><h3>Appendix B</h3><p>Example of a 96-well plate template.</p><div id="ncipr11.appb.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Legend%3A%20Row%20(A)%2C%20Media%20Negative%20Control%3B%20Row%20(B)%2C%20Samples%3B%20Row%20(C)%2C%20Positive%20Control&p=BOOKS&id=604911_ncipr11appbf1.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/NBK604911/bin/ncipr11appbf1.jpg" alt="Legend: Row (A), Media Negative Control; Row (B), Samples; Row (C), Positive Control" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Legend: Row (A), Media Negative Control; Row (B), Samples; Row (C), Positive Control. Each timepoint will be measured individually, as it is harvested.</p></div></div></div><div id="ncipr11.appc"><h3>Appendix C</h3><p>Example of a 96-well plate template.</p><div id="ncipr11.appc.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Legend%3A%20Yellow%2C%20BSA%20Standards%3B%20Green%2C%20Media%20Controls%3B%20Blue%2C%20Samples%3B%20Pink%2C%20Positive%20Controls&p=BOOKS&id=604911_ncipr11appcf1.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/NBK604911/bin/ncipr11appcf1.jpg" alt="Legend: Yellow, BSA Standards; Green, Media Controls; Blue, Samples; Pink, Positive Controls" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Legend: Yellow, BSA Standards; Green, Media Controls; Blue, Samples; Pink, Positive Controls. Each sample is run in duplicate.</p></div></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="ncipr11.suggestedcitation">Stern, ST, Potter TP, NCL Method GTA-5: LLC-PK1 Kidney Cell Apoptosis 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/E9YS-N327" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/E9YS-N327</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="_NBK604911_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">Timothy M. Potter</span>, B.S.</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, Potter TM. LLC-PK1 Kidney Cell Apoptosis 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-5.<span class="bk_cite_avail"></span> doi: 10.17917/E9YS-N327</p></div><div class="small-screen-prev"><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="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/ncipr10/?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="figobncipr11fig1"><div id="ncipr11.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%20LLC-PK1%20Cell%20Culture%20Appearance.&p=BOOKS&id=604911_ncipr11f1.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/NBK604911/bin/ncipr11f1.jpg" alt="Figure 1. Example of LLC-PK1 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 LLC-PK1 Cell Culture Appearance</span></h3><div class="caption"><p>Image was taken with a phase contrast microscope at 225X magnification. LLC-PK1 cells are approximately 80% confluent at this stage.</p></div></div></article><article data-type="fig" id="figobncipr11appafig1"><div id="ncipr11.appa.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=All%20samples%20are%20run%20in%20triplicate&p=BOOKS&id=604911_ncipr11appaf1.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/NBK604911/bin/ncipr11appaf1.jpg" alt="All samples are run in triplicate" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>All samples are run in triplicate. The following timepoints are recommended: 0 hr (media control), 6 hr (sample and media control), 24 hr (sample, positive control, and media control), and 48 hr (sample and media control) in which case 5 plates will be necessary.</p></div></div></article><article data-type="fig" id="figobncipr11appbfig1"><div id="ncipr11.appb.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Legend%3A%20Row%20(A)%2C%20Media%20Negative%20Control%3B%20Row%20(B)%2C%20Samples%3B%20Row%20(C)%2C%20Positive%20Control&p=BOOKS&id=604911_ncipr11appbf1.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/NBK604911/bin/ncipr11appbf1.jpg" alt="Legend: Row (A), Media Negative Control; Row (B), Samples; Row (C), Positive Control" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Legend: Row (A), Media Negative Control; Row (B), Samples; Row (C), Positive Control. Each timepoint will be measured individually, as it is harvested.</p></div></div></article><article data-type="fig" id="figobncipr11appcfig1"><div id="ncipr11.appc.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Legend%3A%20Yellow%2C%20BSA%20Standards%3B%20Green%2C%20Media%20Controls%3B%20Blue%2C%20Samples%3B%20Pink%2C%20Positive%20Controls&p=BOOKS&id=604911_ncipr11appcf1.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/NBK604911/bin/ncipr11appcf1.jpg" alt="Legend: Yellow, BSA Standards; Green, Media Controls; Blue, Samples; Pink, Positive Controls" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Legend: Yellow, BSA Standards; Green, Media Controls; Blue, Samples; Pink, Positive Controls. Each sample is run in duplicate.</p></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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