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<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="Detection of Intracellular Complement Activation in Human T Lymphocytes" /><meta name="citation_publisher" content="National Cancer Institute (US)" /><meta name="citation_date" content="2019/02" /><meta name="citation_author" content="Anna N. Ilinskaya" /><meta name="citation_author" content="Ankit Shah" /><meta name="citation_author" content="Marina A. Dobrovolskaia" /><meta name="citation_pmid" content="39013047" /><meta name="citation_doi" content="10.17917/Y4CQ-SV79" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK604940/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Detection of Intracellular Complement Activation in Human T Lymphocytes" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Cancer Institute (US)" /><meta name="DC.Contributor" content="Anna N. Ilinskaya" /><meta name="DC.Contributor" content="Ankit Shah" /><meta name="DC.Contributor" content="Marina A. Dobrovolskaia" /><meta name="DC.Date" content="2019/02" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK604940/" /><meta name="description" content="The complement present in plasma is a group of approximately 30 proteins which are produced by the liver. Plasma complement plays an important role in innate immunity and promotes adaptive immunity against pathogens. Activation of plasma complement may occur in response to a drug product. Such undesirable activation results in immediate-type hypersensitivity reactions independent of immunoglobulin E. These reactions are often referred to as Complement Activation Related Pseudoallergy (CARPA). Although CARPA has been observed in the clinic with various types of drug products, nanoformulations, and especially PEGylated liposomes, are frequent causes of CARPA in patients [1, 2]." /><meta name="og:title" content="Detection of Intracellular Complement Activation in Human T Lymphocytes" /><meta name="og:type" content="book" /><meta name="og:description" content="The complement present in plasma is a group of approximately 30 proteins which are produced by the liver. Plasma complement plays an important role in innate immunity and promotes adaptive immunity against pathogens. Activation of plasma complement may occur in response to a drug product. Such undesirable activation results in immediate-type hypersensitivity reactions independent of immunoglobulin E. These reactions are often referred to as Complement Activation Related Pseudoallergy (CARPA). Although CARPA has been observed in the clinic with various types of drug products, nanoformulations, and especially PEGylated liposomes, are frequent causes of CARPA in patients [1, 2]." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK604940/" /><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/ncipr25/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK604940/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. doi: 10.17917/Y4CQ-SV79</p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/nciprotocols/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-nciprotocols-lrg.png" alt="Cover of National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK604940_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK604940_dtls__"><div>Bethesda (MD): <a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Cancer Institute (US)</a>; 2005 May 1-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/nciprotocols/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr26/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/nciprotocols/section2/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK604940_"><span class="label">NCL Method ITA-26</span><span class="title" itemprop="name">Detection of Intracellular Complement Activation in Human T Lymphocytes</span></h1><div class="subtitle whole_rhythm">Version 1</div><p class="contrib-group"><span itemprop="author">Anna N. Ilinskaya</span>, Ph.D., <span itemprop="author">Ankit Shah</span>, Ph.D., and <span itemprop="author">Marina A. Dobrovolskaia</span>, Ph.D.</p><a data-jig="ncbitoggler" href="#__NBK604940_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK604940_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Anna N. Ilinskaya</span>, Ph.D., <span itemprop="author">Ankit Shah</span>, Ph.D., and <span itemprop="author">Marina A. Dobrovolskaia</span>, Ph.D.<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>1</sup>.</p><h4>Contact</h4><div class="affiliation"><sup>1</sup> <span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></div><div class="affiliation"><sup>1</sup> Nanotechnology Characterization Lab, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD 21702</div><div><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author.</div></div><p class="small">Published: <span itemprop="datePublished">February 2019</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="ncipr25.s1"><h2 id="_ncipr25_s1_">1. Introduction</h2><p>The complement present in plasma is a group of approximately 30 proteins which are produced by the liver. Plasma complement plays an important role in innate immunity and promotes adaptive immunity against pathogens. Activation of plasma complement may occur in response to a drug product. Such undesirable activation results in immediate-type hypersensitivity reactions independent of immunoglobulin E. These reactions are often referred to as Complement Activation Related Pseudoallergy (CARPA). Although CARPA has been observed in the clinic with various types of drug products, nanoformulations, and especially PEGylated liposomes, are frequent causes of CARPA in patients [<a class="bk_pop" href="#ncipr25.ref1">1</a>, <a class="bk_pop" href="#ncipr25.ref2">2</a>].</p><p>Activation of plasma complement is initiated via three main pathways, the classical, the alternative and the lectin pathway, all of which converge at the complement component 3 (C3) to form a common, terminal pathway leading to the formation of the membrane attack complex [<a class="bk_pop" href="#ncipr25.ref3">3</a>]. Cleavage of C3, C4 and C5 components of the plasma complement results in generation of several split products, C3a, C4a, and C5a, which are also known as anaphylatoxins. The anaphylatoxins are the main triggers of the symptoms observed in CARPA. The larger split products (C3b, C4b, and C5b) are opsonins which bind to and accelerate the clearance of pathogens.</p><p>Recently, the concept of complement has been expanded to include so-called intracellular complement [<a class="bk_pop" href="#ncipr25.ref4">4</a>]. The complement protein C3 has been found in many cells including lymphocytes, monocytes, endothelial and even cancer cells. The activation of intracellular complement generates split products which are exported from the cell onto the membrane. Since the activation of intracellular complement in T-lymphocytes was found to correlate with autoimmune disorders [<a class="bk_pop" href="#ncipr25.ref4">4</a>], and growing evidence is available for the involvement of T-lymphocytes in the development of drug-induced hypersensitivity reactions [<a class="bk_pop" href="#ncipr25.ref5">5</a>], understanding the ability of nanomaterials to activate intracellular complement may aid in establishing long-term safety profiles for these materials.</p></div><div id="ncipr25.s2"><h2 id="_ncipr25_s2_">2. Principles</h2><p>This assay has been adapted from the original study described by Liszewski, et al., [<a class="bk_pop" href="#ncipr25.ref4">4</a>] and optimized to support nanoparticle screening. Human peripheral blood mononuclear cells are treated with nanoparticles and controls, and the presence of the complement split products on the cell surface is then monitored by flow cytometry. Although the gating strategy used in this protocol is specific to CD3-positive lymphocytes (T-cells), this protocol can be adjusted to detect intracellular complement on the surface of other cells. The assay requires 400 µL of the test nanomaterial at a concentration determined through previous cytotoxicity testing of the nanomaterial. The details of nanoparticle concentration selection are provided in <a href="#ncipr25.lt33">section 4.2</a> of the protocol.</p></div><div id="ncipr25.s3"><h2 id="_ncipr25_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="ncipr25.l1" class="temp-labeled-list"><dt>3.1.</dt><dd id="ncipr25.lt1"><p class="no_top_margin">Reagents
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<ol id="ncipr25.l2"><li id="ncipr25.lt2" class="half_rhythm"><div>Human blood coagulated with Li-heparin</div></li><li id="ncipr25.lt3" class="half_rhythm"><div>PBS (HyClone, AQB 22934)</div></li><li id="ncipr25.lt4" class="half_rhythm"><div>Ficoll-Paque Premium (GE HealthCare ,17-5442-03)</div></li><li id="ncipr25.lt5" class="half_rhythm"><div>Fetal bovine serum (Hyclone, SH30070.03)</div></li><li id="ncipr25.lt6" class="half_rhythm"><div>RPMI1640 (Invitrogen, 11875-119)</div></li><li id="ncipr25.lt7" class="half_rhythm"><div>Pen/Strep solution (Invitrogen, 15140-148)</div></li><li id="ncipr25.lt8" class="half_rhythm"><div>Cellometer ViaStain AOPI Staining Solution in PBS (Nexcelom Biosciences, CS2-0106; or other reagent for distinguishing between live and dead cells)</div></li><li id="ncipr25.lt9" class="half_rhythm"><div>Albumin, from bovine serum (BSA) (Sigma, A4503)</div></li><li id="ncipr25.lt10" class="half_rhythm"><div>Purified Mouse Anti-Human CD3 IgG2a (Abcam ab86883; use for positive control)</div></li><li id="ncipr25.lt11" class="half_rhythm"><div>Purified Mouse Anti-Human CD3 IgG2a (eBioscience 16-0037; use flow cytometry to gate CD3 positive cells)</div></li><li id="ncipr25.lt12" class="half_rhythm"><div>Purified Mouse Anti-Human C3adesArg, clone 2991, (NEO-Epitope) IgG1 (Hycult Biotech, HM2074)</div></li><li id="ncipr25.lt13" class="half_rhythm"><div>Purified Mouse IgG1 Isotype control (BD Pharmingen, 554121)</div></li><li id="ncipr25.lt14" class="half_rhythm"><div>Purified Mouse IgG2a Isotype control (BD Pharmigen, 554126)</div></li><li id="ncipr25.lt15" class="half_rhythm"><div>APC Rat Anti-Mouse IgG2a (eBioscience, 17-4210)</div></li><li id="ncipr25.lt16" class="half_rhythm"><div>PE Rat Anti-Mouse IgG1 (eBioscience, 12-4015)</div></li></ol></p></dd><dt>3.2.</dt><dd id="ncipr25.lt17"><p class="no_top_margin">Materials
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<ol id="ncipr25.l3"><li id="ncipr25.lt18" class="half_rhythm"><div>Polystyrene round bottom 12 × 75 mm<sup>2</sup> Falcon tubes (Corning, 352058)</div></li><li id="ncipr25.lt19" class="half_rhythm"><div>Pipettes, 0.05 to 10 mL</div></li><li id="ncipr25.lt20" class="half_rhythm"><div>24-well cell culture plates (Corning, 3524)</div></li><li id="ncipr25.lt21" class="half_rhythm"><div>Polypropylene tubes, 5 mL</div></li></ol></p></dd><dt>3.3.</dt><dd id="ncipr25.lt22"><p class="no_top_margin">Equipment
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<ol id="ncipr25.l4"><li id="ncipr25.lt23" class="half_rhythm"><div>Centrifuge, 400xg (or ~1440 rpm)</div></li><li id="ncipr25.lt24" class="half_rhythm"><div>Refrigerator, 2-8°C</div></li><li id="ncipr25.lt25" class="half_rhythm"><div>Freezer, −20°C</div></li><li id="ncipr25.lt26" class="half_rhythm"><div>Cell culture incubator with 5% CO<sub>2</sub> and 95% humidity</div></li><li id="ncipr25.lt27" class="half_rhythm"><div>Biohazard safety cabinet approved for level II handling of biological material</div></li><li id="ncipr25.lt28" class="half_rhythm"><div>Inverted microscope</div></li><li id="ncipr25.lt29" class="half_rhythm"><div>Vortex</div></li><li id="ncipr25.lt30" class="half_rhythm"><div>Cellometer or hemocytometer</div></li><li id="ncipr25.lt31" class="half_rhythm"><div>Flow Cytometer (e.g., FACSCalibur)</div></li></ol></p></dd></dl></div><div id="ncipr25.s4"><h2 id="_ncipr25_s4_">4. Reagent and Control Preparation</h2><dl id="ncipr25.l5" class="temp-labeled-list"><dt>4.1.</dt><dd id="ncipr25.lt32"><p class="no_top_margin">Complete RPMI</p><p>The complete RPMI medium should contain 10% FBS (heat-inactivated), 4 mM L-glutamine, and 100 U/mL penicillin and 100 µg/mL streptomycin sulphate. Store at 2-8°C protected from light for no longer than 1 month. Before use, warm the medium in a 37°C water bath.</p></dd><dt>4.2.</dt><dd id="ncipr25.lt33"><p class="no_top_margin">Preparation of test nanomaterials</p><p>The important aspect of this test is to assess nanoparticles at non-cytotoxic concentrations, i.e., those not affecting cell viability. Therefore, there is no standard concentration for this test. Each nanomaterial should be evaluated for cytotoxicity before analyzing in this assay. To achieve this, titrate nanoparticles in peripheral blood mononuclear cells (PBMC) starting with the highest reasonable/achievable concentration to select the highest concentration which results in cell viability no less than 80% upon 1 hr incubation with the nanomaterial. Trypan blue exclusion test, MTT, 7-ADD, Cellometer or other relevant cell viability assays can be used for this purpose. Since the dilution factor for this assay is 5, the stock concentration of nanoparticle required for the test should be 5 times higher than this concentration. For example, if the cell viability test determined that the highest non-toxic concentration of nanoparticles in PBMC is 0.1 mg/mL, then you will need 400 µL of a nanoparticle stock at a concentration 0.5 mg/mL. Two hundred (200) µL of this stock will be used directly to treat the cells, and the rest of the material will be used to prepare three serial 1:5 dilutions of this stock. In this example, the final concentrations tested in the assay will be 0.1, 0.02, 0.004 and 0.0008 mg/mL. If a test-nanomaterial is loaded with a cytotoxic drug, the concentrations should reflect the drug and the carrier concentrations. It is also important to include a carrier control.</p></dd><dt>4.3.</dt><dd id="ncipr25.lt34"><p class="no_top_margin">Heat-inactivated fetal bovine serum</p><p>Thaw a bottle of FBS at room temperature or overnight at 2-8°C and allow to equilibrate to room temperature. Incubate 30 minutes in a 56°C water bath, mixing every 5 minutes to heat-inactivate it. Single-use aliquots may be stored at 2-8°C for up to one month or at a nominal temperature of −20°C indefinitely.</p></dd><dt>4.4.</dt><dd id="ncipr25.lt35"><p class="no_top_margin">Staining buffer (0.1% bovine serum albumin (BSA) in PBS)</p><p>Weigh 50 mg of the reagent and add into 50 mL ice-cold 1X PBS. Mix well and place on ice. Prepare fresh before each use. Discard any leftover after experiment is complete.</p></dd><dt>4.5.</dt><dd id="ncipr25.lt36"><p class="no_top_margin">Positive control</p><p>Cells treated for 1 hr with 5 µg/test of Mouse Anti-Human CD3 IgG2a (Abcam cat#ab86883).</p></dd><dt>4.6.</dt><dd id="ncipr25.lt37"><p class="no_top_margin">Negative control</p><p>Use cells without treatment as a negative control.</p></dd></dl><blockquote><p><i>Note: For flow cytometry analysis include a tube with unlabeled cells, which is processed as other tubes except no antibodies added</i>.</p></blockquote></div><div id="ncipr25.s5"><h2 id="_ncipr25_s5_">5. Isolation of Human PBMC</h2><dl id="ncipr25.l6" class="temp-labeled-list"><dt>5.1.</dt><dd id="ncipr25.lt38"><p class="no_top_margin">Place freshly drawn blood into 15 or 50 mL conical centrifuge tubes, add an equal volume of room-temperature PBS and mix well.</p></dd><dt>5.2.</dt><dd id="ncipr25.lt39"><p class="no_top_margin">Slowly layer the Ficoll-Paque solution underneath the blood/PBS mixture by placing the tip of the pipet containing Ficoll-Paque at the bottom of the blood sample tube. Alternatively, blood/PBS mixture may be slowly layered over Ficoll-Paque solution. Use 3 mL of Ficoll-Paque solution per 4 mL of blood/PBS mixture.
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<blockquote><p><i>Note: to maintain Ficoll-blood interface it is helpful to hold the tube at a 45</i>° <i>angle</i>.</p></blockquote></p></dd><dt>5.3.</dt><dd id="ncipr25.lt40"><p class="no_top_margin">Centrifuge 30 minutes at 900xg, 18-20°C without brake.</p></dd><dt>5.4.</dt><dd id="ncipr25.lt41"><p class="no_top_margin">Using a sterile pipet, remove the upper layer containing plasma and platelets and discard it.</p></dd><dt>5.5.</dt><dd id="ncipr25.lt42"><p class="no_top_margin">Using a fresh sterile pipet, transfer the mononuclear cell layer into another centrifuge tube.</p></dd><dt>5.6.</dt><dd id="ncipr25.lt43"><p class="no_top_margin">Wash cells by adding an excess of HBSS and centrifuging for 10 min at 400xg, 18-20°C. The HBSS volume should be ~3 times the volume of the mononuclear layer.
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<blockquote><p><i>Note: Typically, 4 mL of blood/PBS mixture results in ~2 mL of the mononuclear layer and requires at least 6 mL of HBSS for wash step. We use 10 mL of HBSS per each 2 mL of cells</i>.</p></blockquote></p></dd><dt>5.7.</dt><dd id="ncipr25.lt44"><p class="no_top_margin">Discard supernatant and repeat wash step one more time.</p></dd><dt>5.8.</dt><dd id="ncipr25.lt45"><p class="no_top_margin">Resuspend cells in complete RPMI1640 medium. Count cells and determine viability using AOPI staining solution. If viability is at least 90%, proceed to the next step.</p></dd></dl></div><div id="ncipr25.s6"><h2 id="_ncipr25_s6_">6. Treatment with Nanoparticles and Controls</h2><dl id="ncipr25.l7" class="temp-labeled-list"><dt>6.1.</dt><dd id="ncipr25.lt46"><p class="no_top_margin">Adjust cell count to1.25×10<sup>6</sup> cells/mL.</p></dd><dt>6.2.</dt><dd id="ncipr25.lt47"><p class="no_top_margin">Aliquot 800 µL of cell suspension prepared in <a href="#ncipr25.lt45">step 5.8</a> per each well of a 24 well-plate.
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<blockquote><p><i>Note: plan additional wells for isotype controls</i>.</p></blockquote></p></dd><dt>6.3.</dt><dd id="ncipr25.lt48"><p class="no_top_margin">Add 200 µL of test-nanoparticles or control to appropriate wells.</p></dd><dt>6.4.</dt><dd id="ncipr25.lt49"><p class="no_top_margin">After 1 hour of incubation with test-materials and controls, harvest the cells and measure cell viability. If viability is ≥80% proceed to the next step. If not, stop the experiment.</p></dd></dl></div><div id="ncipr25.s7"><h2 id="_ncipr25_s7_">7. Preparation of Cells for Flow Cytometry</h2><dl id="ncipr25.l8" class="temp-labeled-list"><dt>7.1.</dt><dd id="ncipr25.lt50"><p class="no_top_margin">Harvest cells into polystyrene round bottom 12 × 75 mm<sup>2</sup> Falcon tubes and centrifuge at 400xg for 5 minutes.</p></dd><dt>7.2.</dt><dd id="ncipr25.lt51"><p class="no_top_margin">Aspirate supernatant and wash cells with 2 mL ice-cold PBS.</p></dd><dt>7.3.</dt><dd id="ncipr25.lt52"><p class="no_top_margin">Resuspend cells with 100 μL of staining buffer.</p></dd><dt>7.4.</dt><dd id="ncipr25.lt53"><p class="no_top_margin">Add 1 μg/test of Purified Mouse Anti-Human C3adesArg (clone 2991)—except for tubes with isotype controls and a tube with unlabeled cells..</p></dd><dt>7.5.</dt><dd id="ncipr25.lt54"><p class="no_top_margin">Add 1 μg/test of Purified Mouse IgG1 and 0.25μg/test of Purified Mouse IgG2a in isotype control #1 and only 1 μg/test of Purified Mouse IgG1 in isotype control #2.
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<blockquote><p><i>Note: It is convenient to prepare an appropriate amount of working solutions of antibodies in staining buffer and add 100 μL of antibodies containing staining solution to the cells</i>.</p></blockquote></p></dd><dt>7.6.</dt><dd id="ncipr25.lt55"><p class="no_top_margin">Add 0.25 μg/test of CD3 (eBioscience) to tubes except PC and isotype control #1 and unlabeled cells.
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<blockquote><p><i>Note: Isotype controls should be tested with the highest concentration of each tested nanoparticle.</i></p></blockquote></p></dd><dt>7.7.</dt><dd id="ncipr25.lt56"><p class="no_top_margin">Incubate on ice for 20 minutes.</p></dd><dt>7.8.</dt><dd id="ncipr25.lt57"><p class="no_top_margin">Wash cells two more times with 1 µL of PBS by centrifugation at 400xg at 4°C for 5 minutes and resuspend cells with 100 µL of ice-cold staining buffer.</p></dd><dt>7.9.</dt><dd id="ncipr25.lt58"><p class="no_top_margin">Add 0.25 µg/test of PE Rat Anti-Mouse IgG1 and 0.5 μg/test APC Rat Anti-Mouse IgG2a to tubes.</p></dd><dt>7.10.</dt><dd id="ncipr25.lt59"><p class="no_top_margin">Incubate at 4°C for 20 minutes.</p></dd><dt>7.11.</dt><dd id="ncipr25.lt60"><p class="no_top_margin">Wash cells twice with 1 mL of PBS by centrifugation at 400xg for 5 minutes.</p></dd><dt>7.12.</dt><dd id="ncipr25.lt61"><p class="no_top_margin">Resuspend with 400 µL of PBS.</p></dd><dt>7.13.</dt><dd id="ncipr25.lt62"><p class="no_top_margin">Test samples using flow cytometer.
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<blockquote><p><i>Note: All staining procedures should be performed on ice, and centrifuge temperature must be adjusted to 4°C</i>.</p></blockquote></p></dd></dl></div><div id="ncipr25.s8"><h2 id="_ncipr25_s8_">8. Flow Cytometry</h2><blockquote><p><i>At the NCL, we use FACSCalibur. The procedure below is based on our experience with this instrument. If you are using another cytometer, please follow the procedure specific to that instrument</i>.</p></blockquote><dl id="ncipr25.l9" class="temp-labeled-list"><dt>8.1.</dt><dd id="ncipr25.lt63"><p class="no_top_margin">Switch on the instrument. Make sure the sheath tank is full and the waste tank is empty.</p></dd><dt>8.2.</dt><dd id="ncipr25.lt64"><p class="no_top_margin">To get consistent results, run BD FACSComp software using BD CaliBRITE beads.</p></dd><dt>8.3.</dt><dd id="ncipr25.lt65"><p class="no_top_margin">Adjust FSC vs. SSC dot plot using unlabeled cells to get the population of cells in the plot. See <a class="figpopup" href="/books/NBK604940/figure/ncipr25.fig1/?report=objectonly" target="object" rid-figpopup="figncipr25fig1" rid-ob="figobncipr25fig1">Figure 1A</a> below for example.</p></dd><dt>8.4.</dt><dd id="ncipr25.lt66"><p class="no_top_margin">Set the threshold to remove most of the debris.</p></dd><dt>8.5.</dt><dd id="ncipr25.lt67"><p class="no_top_margin">Gate lymphocyte population (gate 1). See <a class="figpopup" href="/books/NBK604940/figure/ncipr25.fig1/?report=objectonly" target="object" rid-figpopup="figncipr25fig1" rid-ob="figobncipr25fig1">Figure 1A</a> below for example.</p></dd><dt>8.6.</dt><dd id="ncipr25.lt68"><p class="no_top_margin">Create an FL2 vs. FL4 dot plot. Limit this dot plot to the cell population from gate 1.</p></dd><dt>8.7.</dt><dd id="ncipr25.lt69"><p class="no_top_margin">Adjust FL2 and FL4 settings to get cells in the left low corner of FL2 vs. FL4 dot plot using unlabeled/untreated cells and isotype control #1.</p></dd><dt>8.8.</dt><dd id="ncipr25.lt70"><p class="no_top_margin">Set up quadrant regions based on isotype control #2. See <a class="figpopup" href="/books/NBK604940/figure/ncipr25.fig1/?report=objectonly" target="object" rid-figpopup="figncipr25fig1" rid-ob="figobncipr25fig1">Figure 1B</a> below for example.</p></dd><dt>8.9.</dt><dd id="ncipr25.lt71"><p class="no_top_margin">Acquire and save the data.</p></dd><dt>8.10.</dt><dd id="ncipr25.lt72"><p class="no_top_margin">Follow the instrument closing procedure.</p></dd></dl><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr25fig1" co-legend-rid="figlgndncipr25fig1"><a href="/books/NBK604940/figure/ncipr25.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr25fig1" rid-ob="figobncipr25fig1"><img class="small-thumb" src="/books/NBK604940/bin/ncipr25f1.gif" src-large="/books/NBK604940/bin/ncipr25f1.jpg" alt="Figure 1. Example of Flow Cytometry Analysis of C3a Expression." /></a><div class="icnblk_cntnt" id="figlgndncipr25fig1"><h4 id="ncipr25.fig1"><a href="/books/NBK604940/figure/ncipr25.fig1/?report=objectonly" target="object" rid-ob="figobncipr25fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Example of Flow Cytometry Analysis of C3a Expression. (A) FSC vs. SSC dot plot and gate to select the cell population of interest. (B) Isotype control #2 (FL2 vs. FL4) (C) Negative control (FL2 vs. FL4) (D) Positive control (FL2 vs. FL4). </p></div></div></div><div id="ncipr25.s9"><h2 id="_ncipr25_s9_">9. Data Analysis and Report</h2><p>Use appropriate software to analyze the data acquired in <a href="#ncipr25.lt71">step 8.9</a>. CellQuest or other flow cytometry software can be used. Here, FCSExpress from De Novo Software Solutions, Inc. was used. Report the data as percent double positive cells, which is determined by comparing the number of double positive cells against that in the negative control.</p></div><div id="ncipr25.s10"><h2 id="_ncipr25_s10_">10. Acceptance Criteria</h2><dl id="ncipr25.l10" class="temp-labeled-list"><dt>10.1.</dt><dd id="ncipr25.lt73"><p class="no_top_margin">The run is acceptable if no nonspecific binding is detected in isotype control #1.</p></dd><dt>10.2.</dt><dd id="ncipr25.lt74"><p class="no_top_margin">The run is acceptable if the positive control is positive, i.e., more than 10% of cells are positive in C3adesArg staining.</p></dd></dl></div><div id="ncipr25.rl.r1"><h2 id="_ncipr25_rl_r1_">11. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ncipr25.ref1">Szebeni
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J, Alving
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CR, Rosivall
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L
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et al.: Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles. <em>J Liposome Res</em>
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17(2), 107–117 (2007).
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[<a href="https://pubmed.ncbi.nlm.nih.gov/17613700" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17613700</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ncipr25.ref2">Salvador-Morales
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C, Sim
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RB: <em>Complement activation</em>. In: <em>Handbook of immunological properties of engineered nanomaterials</em>, Dobrovolskaia
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MA, Mcneil
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SE (Eds). World Scientific, Singapore
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357–384 (2013).</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ncipr25.ref3">Sahu
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A, Lambris
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JD: Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. <em>Immunol Rev</em>
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180, 35–48 (2001).
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[<a href="https://pubmed.ncbi.nlm.nih.gov/11414361" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11414361</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ncipr25.ref4">Liszewski
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MK, Kolev
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M, Le Friec
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G
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et al.: Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation. <em>Immunity</em>
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39(6), 1143–1157 (2013).
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[<a href="/pmc/articles/PMC3865363/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3865363</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24315997" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24315997</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ncipr25.ref5">Pavlos
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R, Mallal
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S, Ostrov
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D
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et al.: T Cell-Mediated Hypersensitivity Reactions to Drugs. <em>Annu Rev Med</em>, (2014). [<a href="/pmc/articles/PMC4295772/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4295772</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25386935" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25386935</span></a>]</div></dd></dl></div><div><dl class="temp-labeled-list small"><dt></dt><dd><div><p class="no_top_margin"><p>This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.</p></p></div></dd><dt>*</dt><dd><div id="ncipr25.fn1"><p class="no_top_margin">address correspondence to: <a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></p></div></dd><dt></dt><dd><div><p class="no_top_margin"><div>
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<span class="mixed-citation" id="ncipr25.suggestedcitation">Ilinksaya AN, Shah A, Dobrovolskaia MA, NCL Method ITA-26: Detection of Intracellular Complement Activation in Human T Lymphocytes. <a href="https://ncl.cancer.gov/resources/asaay-cascade-protocols" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://ncl.cancer.gov/resources/asaay-cascade-protocols</a> DOI: <a href="http://dx.crossref.org/10.17917/Y4CQ-SV79" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/Y4CQ-SV79</a></span>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK604940</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/39013047" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">39013047</a></span>DOI: <a href="http://dx.crossref.org/10.17917/Y4CQ-SV79" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/Y4CQ-SV79</a></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr26/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/nciprotocols/section2/" title="Next page in this title">Next ></a></div></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK604940/?report=reader">PubReader</a></li><li><a href="/books/NBK604940/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK604940" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK604940" style="display:none" title="Cite this Page"><div class="bk_tt">Ilinskaya AN, Shah A, Dobrovolskaia MA. Detection of Intracellular Complement Activation in Human T Lymphocytes: Version 1. 2019 Feb. In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method ITA-26.<span class="bk_cite_avail"></span> doi: 10.17917/Y4CQ-SV79</div></div></li><li><a href="/books/NBK604940/pdf/Bookshelf_NBK604940.pdf">PDF version of this page</a> (568K)</li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ncipr25.s1" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ncipr25.s2" ref="log$=inpage&link_id=inpage">Principles</a></li><li><a href="#ncipr25.s3" ref="log$=inpage&link_id=inpage">Reagents, Materials, and Equipment</a></li><li><a href="#ncipr25.s4" ref="log$=inpage&link_id=inpage">Reagent and Control Preparation</a></li><li><a href="#ncipr25.s5" ref="log$=inpage&link_id=inpage">Isolation of Human PBMC</a></li><li><a href="#ncipr25.s6" ref="log$=inpage&link_id=inpage">Treatment with Nanoparticles and Controls</a></li><li><a href="#ncipr25.s7" ref="log$=inpage&link_id=inpage">Preparation of Cells for Flow Cytometry</a></li><li><a href="#ncipr25.s8" ref="log$=inpage&link_id=inpage">Flow Cytometry</a></li><li><a href="#ncipr25.s9" ref="log$=inpage&link_id=inpage">Data Analysis and Report</a></li><li><a href="#ncipr25.s10" ref="log$=inpage&link_id=inpage">Acceptance Criteria</a></li><li><a href="#ncipr25.rl.r1" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=5641612" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=5641612" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/38506996" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Detection of Intracellular Complement Activation by Nanoparticles in Human T Lymphocytes.</a><span class="source">[Methods Mol Biol. 2024]</span><div class="brieflinkpop offscreen_noflow">Detection of Intracellular Complement Activation by Nanoparticles in Human T Lymphocytes.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Ilinskaya A, Shah A, Van Dusen A, Dobrovolskaia MA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Methods Mol Biol. 2024; 2789:109-120. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/18204047" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement.</a><span class="source">[J Biol Chem. 2008]</span><div class="brieflinkpop offscreen_noflow">Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Rawal N, Rajagopalan R, Salvi VP. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Biol Chem. 2008 Mar 21; 283(12):7853-63. Epub 2008 Jan 18.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/6711816" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Radioimmunoassay for anaphylatoxins: a sensitive method for determining complement activation products in biological fluids.</a><span class="source">[Anal Biochem. 1984]</span><div class="brieflinkpop offscreen_noflow">Radioimmunoassay for anaphylatoxins: a sensitive method for determining complement activation products in biological fluids.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wagner JL, Hugli TE. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Anal Biochem. 1984 Jan; 136(1):75-88. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/325806" ref="ordinalpos=1&linkpos=4&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Pathways of complement activation in membranoproliferative glomerulonephritis and allograft rejection.</a><span class="source">[Transplant Proc. 1977]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Pathways of complement activation in membranoproliferative glomerulonephritis and allograft rejection.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Fearon DT, Daha MR, Strom TB, Weiler JM, Carpenter CB, Austen KF. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Transplant Proc. 1977 Mar; 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