122 lines
49 KiB
Text
122 lines
49 KiB
Text
<!DOCTYPE html>
|
|
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" class="no-js no-jr">
|
|
<head>
|
|
<!-- For pinger, set start time and add meta elements. -->
|
|
<script type="text/javascript">var ncbi_startTime = new Date();</script>
|
|
|
|
<!-- Logger begin -->
|
|
<meta name="ncbi_db" content="books">
|
|
<meta name="ncbi_pdid" content="book-part">
|
|
<meta name="ncbi_acc" content="NBK604964">
|
|
<meta name="ncbi_domain" content="nciprotocols">
|
|
<meta name="ncbi_report" content="reader">
|
|
<meta name="ncbi_type" content="fulltext">
|
|
<meta name="ncbi_objectid" content="">
|
|
<meta name="ncbi_pcid" content="/NBK604964/?report=reader">
|
|
<meta name="ncbi_pagename" content="LLC-PK1 Kidney Cytotoxicity Assay - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf">
|
|
<meta name="ncbi_bookparttype" content="chapter">
|
|
<meta name="ncbi_app" content="bookshelf">
|
|
<!-- Logger end -->
|
|
|
|
<!--component id="Page" label="meta"/-->
|
|
<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>LLC-PK1 Kidney Cytotoxicity Assay - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf</title>
|
|
<meta charset="utf-8">
|
|
<meta name="apple-mobile-web-app-capable" content="no">
|
|
<meta name="viewport" content="initial-scale=1,minimum-scale=1,maximum-scale=1,user-scalable=no">
|
|
<meta name="jr-col-layout" content="auto">
|
|
<meta name="jr-prev-unit" content="/books/n/nciprotocols/ncipr78/?report=reader">
|
|
<meta name="jr-next-unit" content="/books/n/nciprotocols/ncipr23/?report=reader">
|
|
<meta name="bk-toc-url" content="/books/n/nciprotocols/?report=toc">
|
|
<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE">
|
|
<meta name="citation_inbook_title" content="National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]">
|
|
<meta name="citation_title" content="LLC-PK1 Kidney Cytotoxicity Assay">
|
|
<meta name="citation_publisher" content="National Cancer Institute (US)">
|
|
<meta name="citation_date" content="2020/04">
|
|
<meta name="citation_author" content="Stephan T. Stern">
|
|
<meta name="citation_author" content="Pavan P. Adiseshaiah">
|
|
<meta name="citation_author" content="Timothy M. Potter">
|
|
<meta name="citation_pmid" content="39013067">
|
|
<meta name="citation_doi" content="10.17917/Q6EC-XC97">
|
|
<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK604964/">
|
|
<link rel="schema.DC" href="http://purl.org/DC/elements/1.0/">
|
|
<meta name="DC.Title" content="LLC-PK1 Kidney Cytotoxicity 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="Pavan P. Adiseshaiah">
|
|
<meta name="DC.Contributor" content="Timothy M. Potter">
|
|
<meta name="DC.Date" content="2020/04">
|
|
<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK604964/">
|
|
<meta name="description" content="This protocol describes the cytotoxicity testing of nanoparticle formulations porcine proximal tubule cells (LLC-PK1), as part of the in vitro NCL preclinical characterization cascade. The protocol utilizes two methods for estimation of cytotoxicity, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release [1–2].">
|
|
<meta name="og:title" content="LLC-PK1 Kidney Cytotoxicity Assay">
|
|
<meta name="og:type" content="book">
|
|
<meta name="og:description" content="This protocol describes the cytotoxicity testing of nanoparticle formulations porcine proximal tubule cells (LLC-PK1), as part of the in vitro NCL preclinical characterization cascade. The protocol utilizes two methods for estimation of cytotoxicity, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release [1–2].">
|
|
<meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK604964/">
|
|
<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/ncipr32/?report=reader">
|
|
<link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK604964/">
|
|
<link href="https://fonts.googleapis.com/css?family=Archivo+Narrow:400,700,400italic,700italic&subset=latin" rel="stylesheet" type="text/css">
|
|
<link rel="stylesheet" href="/corehtml/pmc/jatsreader/ptpmc_3.22/css/libs.min.css">
|
|
<link rel="stylesheet" href="/corehtml/pmc/jatsreader/ptpmc_3.22/css/jr.min.css">
|
|
<meta name="format-detection" content="telephone=no">
|
|
<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">
|
|
<link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_reader.min.css" type="text/css">
|
|
<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>
|
|
|
|
<link rel="shortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico">
|
|
<meta name="ncbi_phid" content="CE8EA3AC7C8EE9010000000000640055.m_5">
|
|
<meta name='referrer' content='origin-when-cross-origin'/><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3852956/3849091.css"></head>
|
|
<body>
|
|
<!-- Book content! -->
|
|
|
|
|
|
<div id="jr" data-jr-path="/corehtml/pmc/jatsreader/ptpmc_3.22/"><div class="jr-unsupported"><table class="modal"><tr><td><span class="attn inline-block"></span><br />Your browser does not support the NLM PubReader view.<br />Go to <a href="/pmc/about/pr-browsers/">this page</a> to see a list of supported browsers<br />or return to the <br /><a href="/books/NBK604964/?report=classic">regular view</a>.</td></tr></table></div><div id="jr-ui" class="hidden"><nav id="jr-head"><div class="flexh tb"><div id="jr-tb1"><a id="jr-links-sw" class="hidden" title="Links"><svg xmlns="http://www.w3.org/2000/svg" version="1.1" x="0px" y="0px" viewBox="0 0 70.6 85.3" style="enable-background:new 0 0 70.6 85.3;vertical-align:middle" xml:space="preserve" width="24" height="24">
|
|
<style type="text/css">.st0{fill:#939598;}</style>
|
|
<g>
|
|
<path class="st0" d="M36,0C12.8,2.2-22.4,14.6,19.6,32.5C40.7,41.4-30.6,14,35.9,9.8"></path>
|
|
<path class="st0" d="M34.5,85.3c23.2-2.2,58.4-14.6,16.4-32.5c-21.1-8.9,50.2,18.5-16.3,22.7"></path>
|
|
<path class="st0" d="M34.7,37.1c66.5-4.2-4.8-31.6,16.3-22.7c42.1,17.9,6.9,30.3-16.4,32.5h1.7c-66.2,4.4,4.8,31.6-16.3,22.7 c-42.1-17.9-6.9-30.3,16.4-32.5"></path>
|
|
</g>
|
|
</svg> Books</a></div><div class="jr-rhead f1 flexh"><div class="head"><a href="/books/n/nciprotocols/ncipr78/?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="body"><div class="t">NCL Method GTA-1, LLC-PK1 Kidney Cytotoxicity Assay: Version 1.3</div><div class="j">National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]</div></div><div class="tail"><a href="/books/n/nciprotocols/ncipr23/?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></div><div id="jr-tb2"><a id="jr-bkhelp-sw" class="btn wsprkl hidden" title="Help with NLM PubReader">?</a><a id="jr-help-sw" class="btn wsprkl hidden" title="Settings and typography in NLM PubReader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512" preserveAspectRatio="none"><path d="M462,283.742v-55.485l-29.981-10.662c-11.431-4.065-20.628-12.794-25.274-24.001 c-0.002-0.004-0.004-0.009-0.006-0.013c-4.659-11.235-4.333-23.918,0.889-34.903l13.653-28.724l-39.234-39.234l-28.72,13.652 c-10.979,5.219-23.68,5.546-34.908,0.889c-0.005-0.002-0.01-0.003-0.014-0.005c-11.215-4.65-19.933-13.834-24-25.273L283.741,50 h-55.484l-10.662,29.981c-4.065,11.431-12.794,20.627-24.001,25.274c-0.005,0.002-0.009,0.004-0.014,0.005 c-11.235,4.66-23.919,4.333-34.905-0.889l-28.723-13.653l-39.234,39.234l13.653,28.721c5.219,10.979,5.545,23.681,0.889,34.91 c-0.002,0.004-0.004,0.009-0.006,0.013c-4.649,11.214-13.834,19.931-25.271,23.998L50,228.257v55.485l29.98,10.661 c11.431,4.065,20.627,12.794,25.274,24c0.002,0.005,0.003,0.01,0.005,0.014c4.66,11.236,4.334,23.921-0.888,34.906l-13.654,28.723 l39.234,39.234l28.721-13.652c10.979-5.219,23.681-5.546,34.909-0.889c0.005,0.002,0.01,0.004,0.014,0.006 c11.214,4.649,19.93,13.833,23.998,25.271L228.257,462h55.484l10.595-29.79c4.103-11.538,12.908-20.824,24.216-25.525 c0.005-0.002,0.009-0.004,0.014-0.006c11.127-4.628,23.694-4.311,34.578,0.863l28.902,13.738l39.234-39.234l-13.66-28.737 c-5.214-10.969-5.539-23.659-0.886-34.877c0.002-0.005,0.004-0.009,0.006-0.014c4.654-11.225,13.848-19.949,25.297-24.021 L462,283.742z M256,331.546c-41.724,0-75.548-33.823-75.548-75.546s33.824-75.547,75.548-75.547 c41.723,0,75.546,33.824,75.546,75.547S297.723,331.546,256,331.546z"></path></svg></a><a id="jr-fip-sw" class="btn wsprkl hidden" title="Find"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 550 600" preserveAspectRatio="none"><path fill="none" stroke="#000" stroke-width="36" stroke-linecap="round" style="fill:#FFF" d="m320,350a153,153 0 1,0-2,2l170,170m-91-117 110,110-26,26-110-110"></path></svg></a><a id="jr-rtoc-sw" class="btn wsprkl hidden" title="Table of Contents"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M20,20h10v8H20V20zM36,20h44v8H36V20zM20,37.33h10v8H20V37.33zM36,37.33h44v8H36V37.33zM20,54.66h10v8H20V54.66zM36,54.66h44v8H36V54.66zM20,72h10v8 H20V72zM36,72h44v8H36V72z"></path></svg></a></div></div></nav><nav id="jr-dash" class="noselect"><nav id="jr-dash" class="noselect"><div id="jr-pi" class="hidden"><a id="jr-pi-prev" class="hidden" title="Previous page"><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 class="pginfo">Page <i class="jr-pg-pn">0</i> of <i class="jr-pg-lp">0</i></div><a id="jr-pi-next" class="hidden" title="Next page"><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><div id="jr-is-tb"><a id="jr-is-sw" class="btn wsprkl hidden" title="Switch between Figures/Tables strip and Progress bar"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><rect x="10" y="40" width="20" height="20"></rect><rect x="40" y="40" width="20" height="20"></rect><rect x="70" y="40" width="20" height="20"></rect></svg></a></div><nav id="jr-istrip" class="istrip hidden"><a id="jr-is-prev" href="#" class="hidden" title="Previous"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M80,40 60,65 80,90 70,90 50,65 70,40z M50,40 30,65 50,90 40,90 20,65 40,40z"></path><text x="35" y="25" textLength="60" style="font-size:25px">Prev</text></svg></a><a id="jr-is-next" href="#" class="hidden" title="Next"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M20,40 40,65 20,90 30,90 50,65 30,40z M50,40 70,65 50,90 60,90 80,65 60,40z"></path><text x="15" y="25" textLength="60" style="font-size:25px">Next</text></svg></a></nav><nav id="jr-progress"></nav></nav></nav><aside id="jr-links-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">NCBI Bookshelf</div></div><div class="cnt lol f1"><a href="/books/">Home</a><a href="/books/browse/">Browse All Titles</a><a class="btn share" target="_blank" rel="noopener noreferrer" href="https://www.facebook.com/sharer/sharer.php?u=https://www.ncbi.nlm.nih.gov/books/NBK604964/"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 33 33" style="vertical-align:middle" width="24" height="24" preserveAspectRatio="none"><g><path d="M 17.996,32L 12,32 L 12,16 l-4,0 l0-5.514 l 4-0.002l-0.006-3.248C 11.993,2.737, 13.213,0, 18.512,0l 4.412,0 l0,5.515 l-2.757,0 c-2.063,0-2.163,0.77-2.163,2.209l-0.008,2.76l 4.959,0 l-0.585,5.514L 18,16L 17.996,32z"></path></g></svg> Share on Facebook</a><a class="btn share" target="_blank" rel="noopener noreferrer" href="https://twitter.com/intent/tweet?url=https://www.ncbi.nlm.nih.gov/books/NBK604964/&text=LLC-PK1%20Kidney%20Cytotoxicity%20Assay"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 33 33" style="vertical-align:middle" width="24" height="24"><g><path d="M 32,6.076c-1.177,0.522-2.443,0.875-3.771,1.034c 1.355-0.813, 2.396-2.099, 2.887-3.632 c-1.269,0.752-2.674,1.299-4.169,1.593c-1.198-1.276-2.904-2.073-4.792-2.073c-3.626,0-6.565,2.939-6.565,6.565 c0,0.515, 0.058,1.016, 0.17,1.496c-5.456-0.274-10.294-2.888-13.532-6.86c-0.565,0.97-0.889,2.097-0.889,3.301 c0,2.278, 1.159,4.287, 2.921,5.465c-1.076-0.034-2.088-0.329-2.974-0.821c-0.001,0.027-0.001,0.055-0.001,0.083 c0,3.181, 2.263,5.834, 5.266,6.438c-0.551,0.15-1.131,0.23-1.73,0.23c-0.423,0-0.834-0.041-1.235-0.118 c 0.836,2.608, 3.26,4.506, 6.133,4.559c-2.247,1.761-5.078,2.81-8.154,2.81c-0.53,0-1.052-0.031-1.566-0.092 c 2.905,1.863, 6.356,2.95, 10.064,2.95c 12.076,0, 18.679-10.004, 18.679-18.68c0-0.285-0.006-0.568-0.019-0.849 C 30.007,8.548, 31.12,7.392, 32,6.076z"></path></g></svg> Share on Twitter</a></div></aside><aside id="jr-rtoc-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Table of Content</div></div><div class="cnt lol f1"><a href="/books/n/nciprotocols/?report=reader">Title Information</a><a href="/books/n/nciprotocols/toc/?report=reader">Table of Contents Page</a></div></aside><aside id="jr-help-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Settings</div></div><div class="cnt f1"><div id="jr-typo-p" class="typo"><div><a class="sf btn wsprkl">A-</a><a class="lf btn wsprkl">A+</a></div><div><a class="bcol-auto btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 200 100" preserveAspectRatio="none"><text x="10" y="70" style="font-size:60px;font-family: Trebuchet MS, ArialMT, Arial, sans-serif" textLength="180">AUTO</text></svg></a><a class="bcol-1 btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M15,25 85,25zM15,40 85,40zM15,55 85,55zM15,70 85,70z"></path></svg></a><a class="bcol-2 btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M5,25 45,25z M55,25 95,25zM5,40 45,40z M55,40 95,40zM5,55 45,55z M55,55 95,55zM5,70 45,70z M55,70 95,70z"></path></svg></a></div></div><div class="lol"><a class="" href="/books/NBK604964/?report=classic">Switch to classic view</a><a href="/books/NBK604964/pdf/Bookshelf_NBK604964.pdf">PDF (757K)</a><a href="/books/NBK604964/?report=printable">Print View</a></div></div></aside><aside id="jr-bkhelp-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Help</div></div><div class="cnt f1 lol"><a id="jr-helpobj-sw" data-path="/corehtml/pmc/jatsreader/ptpmc_3.22/" data-href="/corehtml/pmc/jatsreader/ptpmc_3.22/img/bookshelf/help.xml" href="">Help</a><a href="mailto:info@ncbi.nlm.nih.gov?subject=PubReader%20feedback%20%2F%20NBK604964%20%2F%20sid%3ACE8B5AF87C7FFCB1_0191SID%20%2F%20phid%3ACE8EA3AC7C8EE9010000000000640055.4">Send us feedback</a><a id="jr-about-sw" data-path="/corehtml/pmc/jatsreader/ptpmc_3.22/" data-href="/corehtml/pmc/jatsreader/ptpmc_3.22/img/bookshelf/about.xml" href="">About PubReader</a></div></aside><aside id="jr-objectbox" class="thidden hidden"><div class="jr-objectbox-close wsprkl">✘</div><div class="jr-objectbox-inner cnt"><div class="jr-objectbox-drawer"></div></div></aside><nav id="jr-pm-left" class="hidden"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 40 800" preserveAspectRatio="none"><text font-stretch="ultra-condensed" x="800" y="-15" text-anchor="end" transform="rotate(90)" font-size="18" letter-spacing=".1em">Previous Page</text></svg></nav><nav id="jr-pm-right" class="hidden"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 40 800" preserveAspectRatio="none"><text font-stretch="ultra-condensed" x="800" y="-15" text-anchor="end" transform="rotate(90)" font-size="18" letter-spacing=".1em">Next Page</text></svg></nav><nav id="jr-fip" class="hidden"><nav id="jr-fip-term-p"><input type="search" placeholder="search this page" id="jr-fip-term" autocorrect="off" autocomplete="off" /><a id="jr-fip-mg" class="wsprkl btn" title="Find"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 550 600" preserveAspectRatio="none"><path fill="none" stroke="#000" stroke-width="36" stroke-linecap="round" style="fill:#FFF" d="m320,350a153,153 0 1,0-2,2l170,170m-91-117 110,110-26,26-110-110"></path></svg></a><a id="jr-fip-done" class="wsprkl btn" title="Dismiss find">✘</a></nav><nav 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="_NBK604964_"><span class="label">NCL Method GTA-1</span><span class="title" itemprop="name">LLC-PK1 Kidney Cytotoxicity Assay</span></h1><div class="subtitle whole_rhythm">Version 1.3</div><p class="contribs">Stern ST, Adiseshaiah PP, Potter TM.</p><p class="fm-aai"><a href="#_NBK604964_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="ncipr32.s1"><h2 id="_ncipr32_s1_">1. Introduction</h2><p>This protocol describes the cytotoxicity testing of nanoparticle formulations porcine proximal tubule cells (LLC-PK1), as part of the in vitro NCL preclinical characterization cascade. The protocol utilizes two methods for estimation of cytotoxicity, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release [<a class="bibr" href="#ncipr32.ref1" rid="ncipr32.ref1">1</a>–<a class="bibr" href="#ncipr32.ref2" rid="ncipr32.ref2">2</a>].</p></div><div id="ncipr32.s2"><h2 id="_ncipr32_s2_">2. Principles</h2><ol id="ncipr32.l1"><li id="ncipr32.lt1" class="half_rhythm"><div class="half_rhythm">MTT Assay</div><div class="half_rhythm">MTT is a yellow, water-soluble tetrazolium dye that is reduced by live cells to a water-insoluble, purple formazan. The amount of formazan can be determined by solubilizing it in DMSO and measuring it spectrophotometrically. Comparisons between the spectra of treated and untreated cells can give a relative estimation of cytotoxicity [<a class="bibr" href="#ncipr32.ref3" rid="ncipr32.ref3">3</a>].</div></li><li id="ncipr32.lt2" class="half_rhythm"><div class="half_rhythm">LDH Assay</div><div class="half_rhythm">LDH is a cytoplasmic enzyme that is released into the cytoplasm upon cell lysis. The LDH assay, therefore, is a measure of membrane integrity. The basis of the LDH assay: a) LDH oxidizes lactate to pyruvate, b) pyruvate reacts with the tetrazolium salt INT to form formazan, and c) the water-soluble formazan dye is detected spectrophotometrically [<a class="bibr" href="#ncipr32.ref4" rid="ncipr32.ref4">4</a>, <a class="bibr" href="#ncipr32.ref5" rid="ncipr32.ref5">5</a>].</div></li></ol></div><div id="ncipr32.s3"><h2 id="_ncipr32_s3_">3. Reagents, Materials, 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="ncipr32.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr32.lt3"><p class="no_top_margin">Reagents
|
|
<ol id="ncipr32.l3"><li id="ncipr32.lt4" class="half_rhythm"><div>LLC-PK1 (pig kidney cells) (ATCC, CL-101)</div></li><li id="ncipr32.lt5" class="half_rhythm"><div>MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma, M5655)</div></li><li id="ncipr32.lt6" class="half_rhythm"><div>Paclitaxel injection, USP (NDC 61703-642-09)</div></li><li id="ncipr32.lt7" class="half_rhythm"><div>Dimethyl sulfoxide (Sigma, D5879)</div></li><li id="ncipr32.lt8" class="half_rhythm"><div>Glycine (Sigma, G7126)</div></li><li id="ncipr32.lt9" class="half_rhythm"><div>Sodium chloride (Sigma, S7653)</div></li><li id="ncipr32.lt10" class="half_rhythm"><div>10% Triton-X-100 (Sigma, 93443)</div></li><li id="ncipr32.lt11" class="half_rhythm"><div>M199 Cell culture media (Cambrex, 12-109-F)</div></li><li id="ncipr32.lt12" class="half_rhythm"><div>Fetal bovine serum (Hyclone, SH30070.03)</div></li><li id="ncipr32.lt13" class="half_rhythm"><div>Biovision LDH-cytotoxicity assay kit (Biovision, K311-400)</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr32.lt14"><p class="no_top_margin">Materials
|
|
<ol id="ncipr32.l4"><li id="ncipr32.lt15" class="half_rhythm"><div>Costar 96 well flat bottom cell culture plates (3598)</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr32.lt16"><p class="no_top_margin">Equipment
|
|
<ol id="ncipr32.l5"><li id="ncipr32.lt17" class="half_rhythm"><div>Plate reader (Safire2 –Tecan or equivalent)</div></li><li id="ncipr32.lt18" class="half_rhythm"><div>Centrifuge, 700-800xg with 96-well plate adapter (Allegra X-15R, Beckman Coulter)</div></li><li id="ncipr32.lt19" class="half_rhythm"><div>Orbital plate shaker</div></li><li id="ncipr32.lt20" class="half_rhythm"><div>Incubator, 37˚C with 5% CO2 and 95% humidity</div></li></ol></p></dd></dl></dl></div><div id="ncipr32.s4"><h2 id="_ncipr32_s4_">4. Reagent and Control Preparation</h2><dl id="ncipr32.l6" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr32.lt21"><p class="no_top_margin">Positive Control
|
|
<ol id="ncipr32.l7"><li id="ncipr32.lt22" class="half_rhythm"><div class="half_rhythm">Paclitaxel Injection</div><div class="half_rhythm">Paclitaxel injection stock is 6 mg/mL (MW 853.9) or 7 mM. Prepare an interim stock of 7 µM by adding 10 µL of paclitaxel injection to 9.990 mL of Medium199 with 3% FBS. Dilute to a final test concentration of 125 nM by adding 357 µL of 7 µM paclitaxel injection to 19.643 mL M199 Medium with 3% FBS. Sterile filtration is not required if the paclitaxel injection is sterile prior to dilution.</div></li><li id="ncipr32.lt23" class="half_rhythm"><div class="half_rhythm">0.1% Triton-X-100</div><div class="half_rhythm">Prepare a 20X stock (2%) of Triton X-100 by adding 2 mL of Triton-X-100 to 8 mL.</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>4.2.</dt><dd id="ncipr32.lt24"><p class="no_top_margin">MTT Assay
|
|
<ol id="ncipr32.l8"><li id="ncipr32.lt25" class="half_rhythm"><div>MTT Solution</div><div>Prepare 5 mg/mL MTT in PBS. Store for up to one month at 4˚C in the dark.</div></li><li id="ncipr32.lt26" class="half_rhythm"><div>Glycine Buffer</div><div>Prepare 0.1 M glycine (MW 75.07) with 0.1 M NaCl (MW 58.44), pH 10.5. Store at room temperature.</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>4.3.</dt><dd id="ncipr32.lt27"><p class="no_top_margin">LDH Assay
|
|
<ol id="ncipr32.l9"><li id="ncipr32.lt28" class="half_rhythm"><div>Reconstitute catalyst in 1 mL dH<sub>2</sub>O for 10 min with occasional vortexing. Stable for 2 weeks at 4˚C.</div></li><li id="ncipr32.lt29" class="half_rhythm"><div>Reaction mixture (for one 96-well plate): Add 250 µL of reconstituted catalyst solution to 11.25 mL of dye solution. Once thawed, the kit components are stable for 2 weeks stored at 4C. Reconstituted catalyst solution should be added to the dye solution immediately before use.</div></li></ol></p></dd></dl></dl></div><div id="ncipr32.s5"><h2 id="_ncipr32_s5_">5. Experimental Procedure</h2><dl id="ncipr32.l10" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.</dt><dd id="ncipr32.lt30"><p class="no_top_margin">Cell Preparation (or as recommended by supplier)
|
|
<ol id="ncipr32.l11"><li id="ncipr32.lt31" class="half_rhythm"><div>Harvest cryopreserved cells from prepared flasks. <b>Note</b>: Limit to 20 passages.</div></li><li id="ncipr32.lt32" class="half_rhythm"><div>Count cell concentration using a coulter counter or hemocytometer.</div></li><li id="ncipr32.lt33" class="half_rhythm"><div>Dilute cells to a density of 2.5 × 10<sup>5</sup> cells/mL in M199 (3% FBS) cell culture media.</div></li><li id="ncipr32.lt34" class="half_rhythm"><div>Plate 100 µL cells/well as per plate format (<a href="#ncipr32.app">Appendix</a>) for four 96-well plates (time zero, 4, 24, and 48 hr sample exposure). The format indicates no cells in rows D and E as they serve as particle blanks to be subtracted from cell treatment wells. Each plate accommodates two samples (Rows A–C and F–H). Each nanoparticle is tested at nine dilutions. Column 11 receives the paclitaxel injection positive control and column 12 receives Triton X-100 at the end of the relevant time point.</div></li><li id="ncipr32.lt35" class="half_rhythm"><div>Incubate plates for 24 hr at 5% CO<sub>2</sub>, 37˚C and 95% humidity. Cells are grown to approximately 80% confluence (<a class="figpopup" href="/books/NBK604964/figure/ncipr32.fig1/?report=objectonly" target="object" rid-figpopup="figncipr32fig1" rid-ob="figobncipr32fig1">Figure 1</a>).</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>5.2.</dt><dd id="ncipr32.lt36"><p class="no_top_margin">Time Zero Plate (MTT Assay)
|
|
<ol id="ncipr32.l12"><li id="ncipr32.lt37" class="half_rhythm"><div>Remove time zero plate from incubator and add 10 µL of 20X Triton-X to positive control wells (see <a class="figpopup" href="/books/NBK604964/figure/ncipr32.app.fig1/?report=objectonly" target="object" rid-figpopup="figncipr32appfig1" rid-ob="figobncipr32appfig1">plate</a> format in <a href="#ncipr32.app">Appendix</a>) for a final concentration of 0.1% Triton X-100 (<a href="#ncipr32.lt23">step 4.1.2</a>). Add 100 µL media to all wells. Let the plate set for 10 minutes at room temperature.</div></li><li id="ncipr32.lt38" class="half_rhythm"><div>Remove 100 µL of media from each well and transfer it to another plate, maintaining plate format. Use this plate immediately for the LDH assay (see <a href="#ncipr32.lt62">Section 5.5</a>).</div></li><li id="ncipr32.lt39" class="half_rhythm"><div>Remove remaining media from original plate and discard.</div></li><li id="ncipr32.lt40" class="half_rhythm"><div>Add 200 µL fresh media to all wells.</div></li><li id="ncipr32.lt41" class="half_rhythm"><div>Add 50 µL MTT (<a href="#ncipr32.lt25">step 4.2.1</a>) to all wells.</div></li><li id="ncipr32.lt42" class="half_rhythm"><div>Cover with aluminum foil and incubate at 37˚C for 4 hr.</div></li><li id="ncipr32.lt43" class="half_rhythm"><div>Aspirate media.</div></li><li id="ncipr32.lt44" class="half_rhythm"><div>Add 200 µL DMSO to all wells to solubilize the MTT formazan crystals.</div></li><li id="ncipr32.lt45" class="half_rhythm"><div>Add 25 µL glycine buffer (<a href="#ncipr32.lt26">step 4.2.2</a>) to all wells. Place on shaker to mix.</div></li><li id="ncipr32.lt46" class="half_rhythm"><div>Read absorbance at 570 nm on plate reader using a reference wavelength of 680 nm.</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>5.3.</dt><dd id="ncipr32.lt47"><p class="no_top_margin">Test Sample and Positive Control Addition
|
|
<ol id="ncipr32.l13"><li id="ncipr32.lt48" class="half_rhythm"><div>The highest concentration of nanoparticle tested should be at the limit of solubility or determined by the potency of the test material.</div></li><li id="ncipr32.lt49" class="half_rhythm"><div>Dilute test compound in media, making a total of nine 1:4 dilutions at 2X the desired final concentration. Different dilution series (i.e., 1:2, 1:10) can be done depending on test material potency and availability.</div></li><li id="ncipr32.lt50" class="half_rhythm"><div>Add 100 µL of each sample dilution and positive control to 4, 24 and 48 hr exposure plates as per the plate format (<a href="#ncipr32.app">Appendix</a>), and place in 37˚C incubator with 5% CO<sub>2</sub> and 95% humidity for indicated time. Alternatively, sample and positive control can be made at desired final concentration. Media is aspirated from wells on the test plates, and sample and positive control are add at 200 µL per well as per the plate format (<a href="#ncipr32.app">Appendix</a>).</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>5.4.</dt><dd id="ncipr32.lt51"><p class="no_top_margin">Test Plates, 4, 24 and 48 hr Exposures (MTT Assay)
|
|
<ol id="ncipr32.l14"><li id="ncipr32.lt52" class="half_rhythm"><div>Remove plate from incubator and add 10 µL of 20X Triton-X to positive control wells (see <a class="figpopup" href="/books/NBK604964/figure/ncipr32.app.fig1/?report=objectonly" target="object" rid-figpopup="figncipr32appfig1" rid-ob="figobncipr32appfig1">plate</a> format in <a href="#ncipr32.app">Appendix</a>) for a final concentration of 0.1% Triton X-100 (<a href="#ncipr32.lt23">step 4.1.2</a>). Let the plate set for 10 minutes at room temperature.</div></li><li id="ncipr32.lt53" class="half_rhythm"><div>Remove 100 µL of media from each well and transfer it to another plate, maintaining plate format. Use this plate immediately for the LDH assay (see <a href="#ncipr32.lt62">Section 5.5</a>).</div></li><li id="ncipr32.lt54" class="half_rhythm"><div>Remove remaining media from original plate and discard.</div></li><li id="ncipr32.lt55" class="half_rhythm"><div>Add 200 µL fresh media to all wells.</div></li><li id="ncipr32.lt56" class="half_rhythm"><div>Add 50 µL MTT to all wells.</div></li><li id="ncipr32.lt57" class="half_rhythm"><div>Cover with aluminum foil and incubate for 37˚C for 4 hr.</div></li><li id="ncipr32.lt58" class="half_rhythm"><div>Aspirate media.</div></li><li id="ncipr32.lt59" class="half_rhythm"><div>Add 200 µL of DMSO to each well.</div></li><li id="ncipr32.lt60" class="half_rhythm"><div>Add 25 µL of glycine buffer to each well. Place on shaker to mix.</div></li><li id="ncipr32.lt61" class="half_rhythm"><div>Read absorbance at 570 nm on plate reader using a reference wavelength of 680 nm.</div></li></ol></p></dd></dl><dl class="bkr_refwrap"><dt>5.5.</dt><dd id="ncipr32.lt62"><p class="no_top_margin"><b>Test Plates, 0, 4, 24 and 48 hr Exposures (LDH Assay)</b>
|
|
<blockquote><p><i>(Adapted from Biovision LDH Cytotoxicity Assay Kit, K311-400)</i></p></blockquote>
|
|
<ol id="ncipr32.l15"><li id="ncipr32.lt63" class="half_rhythm"><div>Add 100 µL of the Reaction Mixture (<a href="#ncipr32.lt29">step 4.3.2</a>) to each well of transfer plate.</div><div>Shake plate on an orbital shaker briefly to mix samples.</div></li><li id="ncipr32.lt64" class="half_rhythm"><div>Incubate at room temperature for up to 20 minutes in the dark.</div></li><li id="ncipr32.lt65" class="half_rhythm"><div>Read the plate on plate reader at 490 nm using a reference wavelength of 680 nm.</div></li></ol></p></dd></dl></dl><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr32fig1" co-legend-rid="figlgndncipr32fig1"><a href="/books/NBK604964/figure/ncipr32.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr32fig1" rid-ob="figobncipr32fig1"><img class="small-thumb" src="/books/NBK604964/bin/ncipr32f1.gif" src-large="/books/NBK604964/bin/ncipr32f1.jpg" alt="Figure 1. LLC-PK1 Cell Culture." /></a><div class="icnblk_cntnt" id="figlgndncipr32fig1"><h4 id="ncipr32.fig1"><a href="/books/NBK604964/figure/ncipr32.fig1/?report=objectonly" target="object" rid-ob="figobncipr32fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">LLC-PK1 Cell Culture. 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="ncipr32.s6"><h2 id="_ncipr32_s6_">6. Calculations</h2><p>All samples, positive, negative, and media controls are run in triplicate (e.g., rows A-C or F-H). Each well will be subtracted from its respective cell-free blank (e.g., A2-D2 or C3-D3) in the following calculations. The average of these three values should be used in the equations below for the positive and negative controls (e.g., [(A1-D1) + (B1-D1) + (C1-D1)]/3 = mean media control absorbance for sample 1, or [(A12-D12) + (B12-D12) + (C12-D12)]/3 = mean TritonX positive control absorbance for sample 1. Sample 2 uses the blank row E for subtractions.).</p><dl id="ncipr32.l16" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>6.1.</dt><dd id="ncipr32.lt66"><p class="no_top_margin">MTT Assay
|
|
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr32.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr32.eq1" display="block"><mrow><mi>%</mi><mtext> </mtext><mi>C</mi><mi>e</mi><mi>l</mi><mi>l</mi><mtext> </mtext><mi>V</mi><mi>i</mi><mi>a</mi><mi>b</mi><mi>i</mi><mi>l</mi><mi>i</mi><mi>t</mi><mi>y</mi><mo>=</mo><mfrac><mrow><mo stretchy="false">(</mo><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>a</mi><mi>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</mi><mi>n</mi><mi>c</mi><mi>e</mi><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>c</mi><mi>e</mi><mi>l</mi><mi>l</mi><mtext> </mtext><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi><mtext> </mtext><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>b</mi><mi>l</mi><mi>a</mi><mi>c</mi><mi>k</mi><mo stretchy="false">)</mo></mrow><mrow><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</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>a</mi><mi>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</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></p></dd></dl><dl class="bkr_refwrap"><dt>6.2.</dt><dd id="ncipr32.lt67"><p class="no_top_margin">LDH Assay
|
|
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr32.deq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr32.eq2" display="block"><mrow><mi>%</mi><mtext> </mtext><mi>T</mi><mi>o</mi><mi>t</mi><mi>a</mi><mi>l</mi><mtext> </mtext><mi>L</mi><mi>D</mi><mi>H</mi><mtext> </mtext><mi>L</mi><mi>e</mi><mi>a</mi><mi>k</mi><mi>a</mi><mi>g</mi><mi>e</mi><mo>=</mo><mfrac><mrow><mo stretchy="false">(</mo><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>a</mi><mi>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</mi><mi>n</mi><mi>c</mi><mi>e</mi><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>c</mi><mi>e</mi><mi>l</mi><mi>l</mi><mtext> </mtext><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi><mtext> </mtext><mi>s</mi><mi>a</mi><mi>m</mi><mi>p</mi><mi>l</mi><mi>e</mi><mtext> </mtext><mi>b</mi><mi>l</mi><mi>a</mi><mi>c</mi><mi>k</mi><mo stretchy="false">)</mo><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</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>a</mi><mi>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</mi><mi>n</mi><mi>c</mi><mi>e</mi></mrow><mrow><mo stretchy="false">(</mo><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>T</mi><mi>r</mi><mi>i</mi><mi>t</mi><mi>o</mi><mi>n</mi><mi>X</mi><mtext> </mtext><mi>p</mi><mi>o</mi><mi>s</mi><mi>i</mi><mi>t</mi><mi>i</mi><mi>v</mi><mi>e</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>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</mi><mi>n</mi><mi>c</mi><mi>e</mi><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</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>a</mi><mi>b</mi><mi>s</mi><mi>o</mi><mi>r</mi><mi>b</mi><mi>a</mi><mi>n</mi><mi>c</mi><mi>e</mi><mo stretchy="false">)</mo></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></p></dd></dl><dl class="bkr_refwrap"><dt>6.3.</dt><dd id="ncipr32.lt68"><p class="no_top_margin">Mean, SD and %CV should also be calculated for each positive control, negative control and unknown sample.</p></dd></dl></dl></div><div id="ncipr32.s7"><h2 id="_ncipr32_s7_">7. Acceptance Criteria</h2><ol id="ncipr32.l17"><li id="ncipr32.lt69" class="half_rhythm"><div>The 48 hr % cell viability and % total LDH leakage for the paclitaxel injection positive control should be less than 50% and greater than 25%, respectively.</div></li><li id="ncipr32.lt70" class="half_rhythm"><div>The positive and sample replicate coefficient of variations should be within 50%.</div></li><li id="ncipr32.lt71" class="half_rhythm"><div>The assay is acceptable if criteria 1 and 2 are met. Otherwise, the assay should be repeated until acceptance criteria are met.</div></li><li id="ncipr32.lt72" class="half_rhythm"><div>If the acceptance criteria are met, determine the highest concentration of the nanoparticulate material that does not interfere with the assay system indicated in rows D and E.</div></li><li id="ncipr32.lt73" class="half_rhythm"><div>The concentration–response curves for the 48 hr MTT and LDH data should be classified as having complete (two observed asymptotes) or incomplete (second asymptote not obtained) curves, single point activity (activity at the highest concentration only), or no activity. For all complete 48 hr concentration–response curves, a nonlinear fit of the sigmoidal hill equation should be performed, and an estimate of potency (EC<sub>50</sub>), efficacy (<i>E</i><sub>max</sub>), minimum response (<i>E</i><sub>0</sub>), and hill slope (γ) from the Hill equation (below) fit should be reported. Any excluded points (excluded by outlier analysis) should also be reported.</div></li></ol><div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr32.deq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
|
|
<math id="ncipr32.eq3" display="block"><mrow><mi>E</mi><mo>=</mo><msub><mi>E</mi><mn>0</mn></msub><mo>+</mo><mo stretchy="false">[</mo><mo stretchy="false">(</mo><msub><mi>E</mi><mrow><mi>m</mi><mi>a</mi><mi>x</mi></mrow></msub><mo>−</mo><msub><mi>E</mi><mn>0</mn></msub><mo stretchy="false">)</mo><mo>∗</mo><mi>C</mi><mi>γ</mi><mo>/</mo><mo stretchy="false">(</mo><mi>E</mi><mi>C</mi><msub><mi>γ</mi><mrow><mn>50</mn></mrow></msub><mo>∗</mo><mi>C</mi><mi>γ</mi><mo stretchy="false">)</mo><mo stretchy="false">]</mo></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></div><div id="ncipr32.rl.r1"><h2 id="_ncipr32_rl_r1_">8. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ncipr32.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="ncipr32.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="ncipr32.ref3">Alley, et al. (1988) <em>Cancer Res</em>. 48:589–601.
|
|
[<a href="https://pubmed.ncbi.nlm.nih.gov/3335022" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3335022</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ncipr32.ref4">Decker, T. & Lohmann-Matthes, M.L. (1988) <em>J. Immunol Methods</em>
|
|
15:61–69. [<a href="https://pubmed.ncbi.nlm.nih.gov/3192948" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3192948</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ncipr32.ref5">Korzeniewski, C. & Callewaert, D.M. (1983) <em>J. Immunol Methods</em>
|
|
64:313–320.
|
|
[<a href="https://pubmed.ncbi.nlm.nih.gov/6199426" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6199426</span></a>]</div></dd></dl></dl></div><div id="ncipr32.s8"><h2 id="_ncipr32_s8_">9. Abbreviations</h2><dl><dt id="ncipr32.abb_DL1_DI1">CV</dt><dd><p>coefficient of variation</p></dd><dt id="ncipr32.abb_DL1_DI2">DMSO</dt><dd><p>dimethyl sulfoxide</p></dd><dt id="ncipr32.abb_DL1_DI3">FBS</dt><dd><p>fetal bovine serum</p></dd><dt id="ncipr32.abb_DL1_DI4">INT</dt><dd><p>2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride</p></dd><dt id="ncipr32.abb_DL1_DI5">LDH</dt><dd><p>lactate dehydrogenase</p></dd><dt id="ncipr32.abb_DL1_DI6">LLC-PK1</dt><dd><p>renal epithelial cell line, porcine kidney</p></dd><dt id="ncipr32.abb_DL1_DI7">MTT</dt><dd><p>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</p></dd><dt id="ncipr32.abb_DL1_DI8">MW</dt><dd><p>molecular weight</p></dd><dt id="ncipr32.abb_DL1_DI9">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr32.abb_DL1_DI10">SD</dt><dd><p>standard deviation</p></dd></dl></div><div id="ncipr32.app"><h2 id="_ncipr32_app_">10. Appendix</h2><div id="ncipr32.app.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Plate%20Map.&p=BOOKS&id=604964_ncipr32appf1.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/NBK604964/bin/ncipr32appf1.jpg" alt="Plate Map." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title">Plate Map</span></h3><div class="caption"><p>TS, test sample; D, dilution</p></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>
|
|
<span class="mixed-citation" id="ncipr32.suggestedcitation">Stern ST, Adiseshaiah PP, Potter TP, NCL Method GTA-1: LLC-PK1 Kidney Cytotoxicity 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/Q6EC-XC97" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/Q6EC-XC97</a></span>
|
|
</div></p></div></dd></dl></dl></div><div style="display:none"><div style="display:none" id="figncipr32appfig1"><img alt="Image ncipr32appf1" src-large="/books/NBK604964/bin/ncipr32appf1.jpg" /></div></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK604964_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., DABT, <span itemprop="author">Pavan P. Adiseshaiah</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 2020</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, Adiseshaiah PP, Potter TM. LLC-PK1 Kidney Cytotoxicity Assay: Version 1.3. 2020 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-1.<span class="bk_cite_avail"></span> doi: 10.17917/Q6EC-XC97</p></div><div class="small-screen-prev"><a href="/books/n/nciprotocols/ncipr78/?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/ncipr23/?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="figobncipr32fig1"><div id="ncipr32.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20LLC-PK1%20Cell%20Culture.&p=BOOKS&id=604964_ncipr32f1.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/NBK604964/bin/ncipr32f1.jpg" alt="Figure 1. LLC-PK1 Cell Culture." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">LLC-PK1 Cell Culture</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="figobncipr32appfig1"><div id="ncipr32.app.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Plate%20Map.&p=BOOKS&id=604964_ncipr32appf1.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/NBK604964/bin/ncipr32appf1.jpg" alt="Plate Map." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title">Plate Map</span></h3><div class="caption"><p>TS, test sample; D, dilution</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>
|
|
|
|
|
|
|
|
|
|
<!-- Book content -->
|
|
|
|
<script type="text/javascript" src="/portal/portal3rc.fcgi/rlib/js/InstrumentNCBIBaseJS/InstrumentPageStarterJS.js"> </script>
|
|
|
|
|
|
<!-- CE8B5AF87C7FFCB1_0191SID /projects/books/PBooks@9.11 portal107 v4.1.r689238 Tue, Oct 22 2024 16:10:51 -->
|
|
<span id="portal-csrf-token" style="display:none" data-token="CE8B5AF87C7FFCB1_0191SID"></span>
|
|
|
|
<script type="text/javascript" src="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/js/3968615.js" snapshot="books"></script></body>
|
|
</html>
|