{"id":1832,"date":"2026-06-27T12:26:18","date_gmt":"2026-06-27T11:26:18","guid":{"rendered":"http:\/\/seamlessbio.de\/?page_id=1832"},"modified":"2026-06-27T12:27:15","modified_gmt":"2026-06-27T11:27:15","slug":"application-cytotoxicity-assay","status":"publish","type":"page","link":"https:\/\/seamlessbio.de\/de\/application-cytotoxicity-assay\/","title":{"rendered":"Application Cytotoxicity Assay"},"content":{"rendered":"\t\t<div data-elementor-type=\"wp-page\" data-elementor-id=\"1832\" class=\"elementor elementor-1832\" data-elementor-post-type=\"page\">\n\t\t\t\t<div class=\"elementor-element elementor-element-6b75082 e-con e-atomic-element e-flexbox-base e-9b725a4 \" data-id=\"6b75082\" data-element_type=\"e-flexbox\" data-e-type=\"e-flexbox\" data-interaction-id=\"6b75082\">\n    \t\t<div class=\"elementor-element elementor-element-86b2475 elementor-widget elementor-widget-html\" data-id=\"86b2475\" data-element_type=\"widget\" data-e-type=\"widget\" data-widget_type=\"html.default\">\n\t\t\t\t\t<!-- YOAST SEO SETTINGS\nSlug:             applications\/cytotoxicity-assays\nFocus Keyword:    cytotoxicity assay ADCC CDC MTT AlamarBlue serum FBS Ultra Low IgG\nSEO Title (EN):   Cytotoxicity Assays \u2014 ADCC, CDC, MTT, AlamarBlue, LDH Serum Guide | SeamlessBio\nMeta Desc (EN):   Complete serum guide for ADCC, CDC, MTT, AlamarBlue, LDH, ELISpot. Why FBS Ultra Low IgG for ADCC, Human Serum AB OTC for CDC. 3 interference mechanisms, 3 protocols. SeamlessBio.\n-->\n<section class=\"sb-app\">\n<style>\n.sb-app,.sb-app *,.sb-app *::before,.sb-app *::after{box-sizing:border-box;}\n.sb-app{background:#fff;padding:70px 40px 90px;font-family:Arial,sans-serif;color:#202020;}\n.sb-app .wrap{max-width:1200px;width:100%;margin:0 auto;}\n.sb-app .bc{display:flex;flex-wrap:wrap;gap:8px;margin:0 0 24px;color:#777;font-size:14px;}\n.sb-app .bc a{color:#18b7b2!important;text-decoration:none!important;font-weight:700;}\n.sb-app .bc strong{color:#111;font-weight:800;}\n.sb-app .hero{padding:64px 56px;margin-bottom:48px;border-radius:30px;background:linear-gradient(135deg,#18b7b2 0%,#65d3cf 52%,#e9fbfa 100%);color:#fff;}\n.sb-app .hero .ey{display:inline-block;margin-bottom:14px;font-size:13px;font-weight:900;text-transform:uppercase;letter-spacing:1.8px;}\n.sb-app .hero h1{margin:0 0 18px;color:#fff;font-size:clamp(26px,3.2vw,40px);line-height:1.1;font-weight:900;letter-spacing:-1px;max-width:840px;}\n.sb-app .hero .sub{max-width:720px;margin:0 0 30px;color:#fff;font-size:17px;line-height:1.65;}\n.sb-app .hbtns{display:flex;flex-wrap:wrap;gap:14px;}\n.sb-app .btn1{display:inline-flex;align-items:center;justify-content:center;min-height:46px;padding:0 28px;border-radius:999px;background:#18b7b2;color:#fff!important;text-decoration:none!important;text-transform:uppercase;font-size:13px;font-weight:900;letter-spacing:.6px;transition:all .25s;}\n.sb-app .btn1:hover{background:#139b97;transform:translateY(-2px);}\n.sb-app .btn2{display:inline-flex;align-items:center;justify-content:center;min-height:46px;padding:0 28px;border-radius:999px;background:transparent;border:2px solid #fff;color:#fff!important;text-decoration:none!important;text-transform:uppercase;font-size:13px;font-weight:900;letter-spacing:.6px;transition:all .25s;}\n.sb-app .btn2:hover{background:#fff;color:#139b97!important;transform:translateY(-2px);}\n.sb-app .warn{background:rgba(245,166,35,.1);border-left:4px solid #f5a623;border-radius:8px;padding:18px 22px;margin-bottom:32px;font-size:14px;color:#444;line-height:1.7;}\n.sb-app .warn strong{color:#111;display:block;margin-bottom:6px;font-size:15px;}\n.sb-app .note{background:rgba(24,183,178,.08);border-left:4px solid #18b7b2;border-radius:8px;padding:18px 22px;margin-bottom:32px;font-size:14px;color:#444;line-height:1.7;}\n.sb-app .note strong{color:#111;display:block;margin-bottom:6px;font-size:15px;}\n.sb-app .sec{margin-bottom:34px;padding:34px;border-radius:26px;background:#fff;border:1px solid #eee;}\n.sb-app .sec h2{margin:0 0 14px;color:#111;font-size:22px;font-weight:900;}\n.sb-app .sec h3{margin:14px 0 10px;color:#111;font-size:17px;font-weight:900;}\n.sb-app .sec p{margin:0 0 12px;color:#555;font-size:15px;line-height:1.7;}\n.sb-app .sec ul,.sb-app .sec ol{margin:0 0 14px 0;padding:0;list-style:none;}\n.sb-app .sec li{position:relative;padding:5px 0 5px 20px;color:#555;font-size:14px;line-height:1.6;}\n.sb-app .sec li::before{content:\"\";position:absolute;left:0;top:13px;width:7px;height:7px;border-radius:50%;background:#18b7b2;}\n.sb-app .sec ol li{counter-increment:item;padding-left:24px;}\n.sb-app .sec ol li::before{content:counter(item);width:18px;height:18px;background:#18b7b2;color:#fff;font-size:10px;font-weight:900;display:flex;align-items:center;justify-content:center;border-radius:50%;top:10px;}\n.sb-app .tw{overflow-x:auto;margin-top:16px;}\n.sb-app table{width:100%;border-collapse:collapse;font-size:14px;min-width:480px;}\n.sb-app thead th{background:#18b7b2;color:#fff;padding:11px 13px;text-align:left;font-weight:700;}\n.sb-app tbody tr:nth-child(even){background:#f5fffe;}\n.sb-app tbody tr:hover{background:#e8f9f8;}\n.sb-app tbody td{padding:10px 13px;vertical-align:top;border-bottom:1px solid #e5e5e5;line-height:1.5;font-size:13px;}\n.sb-app table a{color:#18b7b2;text-decoration:none;font-weight:600;}\n.sb-app .ok{color:#18b7b2;font-weight:700;}\n.sb-app .no{color:#aaa;}\n.sb-app .warn-cell{color:#e07a00;font-weight:600;}\n.sb-app .protocol{background:#f7fbfb;border:1px solid rgba(24,183,178,.18);border-radius:14px;padding:22px 26px;margin-bottom:20px;}\n.sb-app .protocol h4{font-size:15px;font-weight:900;color:#111;margin:0 0 12px;}\n.sb-app .protocol p{font-size:13px;color:#555;line-height:1.7;margin:0;}\n.sb-app .faq{margin-bottom:34px;padding:34px;border-radius:26px;background:#f7fbfb;border:1px solid rgba(24,183,178,.18);}\n.sb-app .faq h2{margin:0 0 20px;color:#111;font-size:22px;font-weight:900;}\n.sb-app .faq-item{border-bottom:1px solid #e0e0e0;padding:18px 0;}\n.sb-app .faq-item:last-child{border-bottom:none;padding-bottom:0;}\n.sb-app .faq-q{font-size:15px;font-weight:900;color:#111;margin-bottom:8px;}\n.sb-app .faq-a{font-size:14px;color:#555;line-height:1.7;}\n.sb-app .related{margin-bottom:34px;padding:28px 34px;border-radius:26px;background:#f7fbfb;border:1px solid rgba(24,183,178,.18);}\n.sb-app .related h3{margin:0 0 16px;color:#111;font-size:18px;font-weight:900;}\n.sb-app .rel-grid{display:grid;grid-template-columns:repeat(2,1fr);gap:12px;}\n.sb-app .rel-grid a{display:flex;align-items:center;min-height:50px;padding:12px 16px;border-radius:12px;background:#fff;border:1px solid #e0e0e0;color:#111!important;text-decoration:none!important;font-size:13px;font-weight:700;transition:all .2s;}\n.sb-app .rel-grid a:hover{border-color:#18b7b2;color:#139b97!important;}\n.sb-app .hub-back{text-align:center;padding:20px 0 0;border-top:1px solid rgba(24,183,178,.15);margin-bottom:16px;}\n.sb-app .hub-back a{color:#18b7b2;font-size:14px;font-weight:700;text-decoration:none;}\n.sb-app .cta{padding:54px 34px;border-radius:30px;background:#f7fbfb;border:1px solid rgba(24,183,178,.18);text-align:center;}\n.sb-app .cta h2{margin:0 0 12px;color:#111;font-size:28px;font-weight:900;}\n.sb-app .cta p{max-width:760px;margin:0 auto 26px;color:#555;font-size:15px;line-height:1.65;}\n.sb-app .ctabtns{display:flex;flex-wrap:wrap;gap:14px;justify-content:center;}\n.sb-app .ctab1,.sb-app .ctab2{display:inline-flex;align-items:center;justify-content:center;min-height:46px;padding:0 28px;border-radius:999px;font-size:13px;font-weight:900;text-transform:uppercase;letter-spacing:.6px;text-decoration:none!important;transition:all .25s;}\n.sb-app .ctab1{background:#18b7b2;color:#fff!important;}\n.sb-app .ctab1:hover{background:#139b97;transform:translateY(-2px);}\n.sb-app .ctab2{background:transparent;border:2px solid #18b7b2;color:#18b7b2!important;}\n.sb-app .ctab2:hover{background:#18b7b2;color:#fff!important;transform:translateY(-2px);}\n@media(max-width:1100px){.sb-app .hero{padding:48px 36px;}.sb-app .rel-grid{grid-template-columns:1fr;}}\n@media(max-width:700px){.sb-app{padding:50px 16px 70px;}.sb-app .hero{padding:40px 24px;border-radius:24px;}.sb-app .hero h1{font-size:25px;}.sb-app .hbtns,.sb-app .ctabtns{flex-direction:column;}.sb-app .btn1,.sb-app .btn2,.sb-app .ctab1,.sb-app .ctab2{width:100%;}.sb-app .sec,.sb-app .faq{padding:20px;}.sb-app .cta{padding:36px 20px;}}\n<\/style>\n<div class=\"wrap\">\n  <div class=\"bc\">\n    <a href=\"http:\/\/seamlessbio.de\/\">Home<\/a><span>\/<\/span>\n    <a href=\"https:\/\/www.seamlessbio.de\/applications\/\">Applications<\/a><span>\/<\/span>\n    <strong>Cytotoxicity Assays<\/strong>\n  <\/div>\n\n  <div class=\"hero\">\n    <span class=\"ey\">SeamlessBio \u2014 ADCC \u00b7 CDC \u00b7 MTT \u00b7 AlamarBlue \u00b7 LDH \u00b7 ELISpot<\/span>\n    <h1>Serum Selection for Cytotoxicity Assays \u2014 ADCC, CDC, MTT, AlamarBlue, LDH &amp; ELISpot<\/h1>\n    <p class=\"sub\">Serum is not a neutral background in cytotoxicity assays. It is the most biologically active variable in your system \u2014 containing complement proteins, immunoglobulins, growth factors, and lipid-binding proteins that interact directly with effector cells, target cells, fluorescent readout reagents, and test compounds. The wrong grade does not add variability. It changes the signal being measured.<\/p>\n    <div class=\"hbtns\">\n      <a href=\"https:\/\/www.seamlessbio.de\/about-us\/contact-imprint\/\" class=\"btn1\">Contact Us<\/a>\n      <a href=\"https:\/\/shop.seamlessbio.de\" class=\"btn2\">View in Shop<\/a>\n    <\/div>\n  <\/div>\n\n  <div class=\"warn\">\n    <strong>\u26a0 Three most common serum-related assay failures:<\/strong>\n    1. <strong>Bovine IgG in standard FBS<\/strong> competes with therapeutic mAbs for Fc\u03b3R on NK cells \u2014 reduces measured ADCC activity.<br>\n    2. <strong>Active complement in non-HI serum<\/strong> causes spontaneous lysis of complement-sensitive cell lines \u2014 inflates background cytotoxicity.<br>\n    3. <strong>FBS and BSA bind fluorescent dyes<\/strong> (AlamarBlue, calcein AM) \u2014 serum concentration must be identical in ALL wells including controls.\n  <\/div>\n\n  <!-- QUICK REFERENCE -->\n  <div class=\"sec\">\n    <h2>Quick Reference \u2014 Serum by Assay Type<\/h2>\n    <div class=\"tw\">\n      <table>\n        <thead><tr><th>Assay Type<\/th><th>Recommended Serum<\/th><th>Standard FBS?<\/th><th>Critical Interference<\/th><\/tr><\/thead>\n        <tbody>\n          <tr>\n            <td><strong>ADCC<\/strong> (NK cell-mediated)<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-ultra-low-igg-low-igg\">FBS Ultra Low IgG &lt;5 \u00b5g\/mL<\/a><\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>Bovine IgG competes with mAb for Fc\u03b3RIII on NK cells<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>CDC \u2014 active complement<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-off-the-clot-type-ab\">Human Serum AB OTC (native)<\/a><\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>Bovine complement \u2260 human complement specificity<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>CDC \u2014 negative control<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-type-ab-male\">Human Serum AB Heat Inactivated<\/a><\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>HI destroys complement \u2014 required paired with native for valid CDC<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>MTT \/ MTS<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Low Endotoxin \u22645 EU\/mL<\/a><\/td>\n            <td class=\"warn-cell\">\u26a0 Only LE<\/td>\n            <td>Endotoxin activates TLR4 \u2192 non-specific growth inhibition<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>AlamarBlue \/ Resazurin<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Low Endotoxin \u22645 EU\/mL<\/a><\/td>\n            <td class=\"warn-cell\">\u26a0 Only LE<\/td>\n            <td>FBS\/BSA quench fluorescence \u2014 identical serum % in ALL wells<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>LDH release<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Low Endotoxin, low haemoglobin<\/a><\/td>\n            <td class=\"warn-cell\">\u26a0 Only low Hgb<\/td>\n            <td>Haemolysis releases endogenous LDH \u2192 elevated background<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>ELISpot<\/strong> (IFN-\u03b3, Granzyme B)<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Very Low Endotoxin \u22641 EU\/mL<\/a> or <a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-type-ab-male\">Human Serum AB HI<\/a><\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>Endotoxin activates monocytes \u2192 non-specific spot background<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>Hepatotoxicity<\/strong> (HepG2, HepaRG)<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Low Endotoxin \u22645 EU\/mL<\/a><\/td>\n            <td class=\"warn-cell\">\u26a0 Only LE<\/td>\n            <td>Endotoxin activates hepatoma innate immune responses<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>3D Spheroid cytotoxicity<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-ultra-low-igg-low-igg\">FBS Ultra Low IgG &lt;5 \u00b5g\/mL<\/a> (2\u20135%)<\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>Bovine IgG interferes with anti-tumour antibody readout on spheroids<\/td>\n          <\/tr>\n          <tr>\n            <td><strong>Neutralisation assay<\/strong><\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Very Low Endotoxin \u22641 EU\/mL<\/a><\/td>\n            <td class=\"no\">\u2717 No<\/td>\n            <td>Complement in non-HI FBS can neutralise enveloped viruses<\/td>\n          <\/tr>\n        <\/tbody>\n      <\/table>\n    <\/div>\n  <\/div>\n\n  <!-- ADCC -->\n  <div class=\"sec\">\n    <h2>ADCC \u2014 Why FBS Ultra Low IgG is Non-Negotiable<\/h2>\n    <p>ADCC measures NK cell-mediated killing of antibody-opsonised target cells via Fc\u03b3RIII (CD16). Standard FBS contains 200\u2013800 \u00b5g\/mL bovine IgG which binds directly to Fc\u03b3RIII \u2014 competing with the therapeutic mAb and reducing measured cytotoxicity. Controls cannot correct for this \u2014 the interference occurs before the readout.<\/p>\n    <div class=\"tw\">\n      <table>\n        <thead><tr><th>Parameter<\/th><th>Recommendation<\/th><th>Note<\/th><\/tr><\/thead>\n        <tbody>\n          <tr><td>Serum grade<\/td><td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-ultra-low-igg-low-igg\">FBS Ultra Low IgG &lt;5 \u00b5g\/mL<\/a><\/td><td>Use in effector cell expansion AND assay medium \u2014 consistent throughout<\/td><\/tr>\n          <tr><td>Concentration<\/td><td>5\u201310%<\/td><td>Identical in all wells including controls<\/td><\/tr>\n          <tr><td>Effector cells<\/td><td>PBMC or purified NK cells<\/td><td>Expand in FBS Ultra Low IgG for \u22655 days before assay<\/td><\/tr>\n          <tr><td>Target cell wash<\/td><td>2\u00d7 wash before assay<\/td><td>Remove residual bovine IgG from target cell medium<\/td><\/tr>\n          <tr><td>Readout<\/td><td>LDH release, calcein AM, or flow (PI)<\/td><td>Spontaneous release control must use identical serum concentration<\/td><\/tr>\n          <tr><td>Typical E:T ratio<\/td><td>10:1 to 50:1<\/td><td>Optimise per cell line and mAb concentration<\/td><\/tr>\n        <\/tbody>\n      <\/table>\n    <\/div>\n  <\/div>\n\n  <!-- CDC -->\n  <div class=\"sec\">\n    <h2>CDC \u2014 Why Human Serum AB OTC is Required<\/h2>\n    <p>CDC measures antibody-mediated activation of the complement cascade leading to MAC formation and target cell lysis. Complement is highly species-specific \u2014 bovine complement has different specificity, activation kinetics, and regulatory protein interactions with human cells. Human Serum AB OTC (native, not heat-inactivated) provides active human complement.<\/p>\n    <div class=\"tw\">\n      <table>\n        <thead><tr><th>Application<\/th><th>Serum<\/th><th>Why<\/th><\/tr><\/thead>\n        <tbody>\n          <tr>\n            <td>CDC assay \u2014 active complement<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-off-the-clot-type-ab\">Human Serum AB OTC (native)<\/a> \u2014 10\u201325%<\/td>\n            <td>Active human complement. Type AB \u2014 no anti-A\/B lysis of target cells.<\/td>\n          <\/tr>\n          <tr>\n            <td>CDC assay \u2014 complement-depleted control<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-type-ab-male\">Human Serum AB HI<\/a> \u2014 same concentration<\/td>\n            <td>56\u00b0C\/30 min destroys complement. Paired native\/HI from same donor pool required for valid comparison.<\/td>\n          <\/tr>\n        <\/tbody>\n      <\/table>\n    <\/div>\n  <\/div>\n\n  <!-- 3 INTERFERENCE MECHANISMS -->\n  <div class=\"sec\">\n    <h2>Three Serum Interference Mechanisms Explained<\/h2>\n    <div class=\"tw\">\n      <table>\n        <thead><tr><th>Mechanism<\/th><th>Affected Assays<\/th><th>Serum Component<\/th><th>Solution<\/th><\/tr><\/thead>\n        <tbody>\n          <tr>\n            <td><strong>Fc Receptor Competition<\/strong><br><small>Bovine IgG occupies Fc\u03b3RI\/II\/III on effector cells before mAb is added<\/small><\/td>\n            <td>ADCC, ADCP, 3D spheroid antibody assays<\/td>\n            <td>IgG (200\u2013800 \u00b5g\/mL in standard FBS)<\/td>\n            <td><a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-ultra-low-igg-low-igg\">FBS Ultra Low IgG &lt;5 \u00b5g\/mL<\/a><\/td>\n          <\/tr>\n          <tr>\n            <td><strong>Complement Activation<\/strong><br><small>Active complement lyses complement-sensitive cells spontaneously \u2014 independent of antibody<\/small><\/td>\n            <td>CDC, ELISpot with PBMC, any assay with lymphoblast or RBC target cells<\/td>\n            <td>Complement proteins C1q\u2013C9 (in non-HI serum)<\/td>\n            <td>Heat inactivated serum (56\u00b0C\/30 min) or <a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-type-ab-male\">Human Serum AB HI<\/a><\/td>\n          <\/tr>\n          <tr>\n            <td><strong>Dye &amp; Compound Binding<\/strong><br><small>Serum proteins bind fluorescent dyes and test compounds \u2014 reduces apparent signal<\/small><\/td>\n            <td>AlamarBlue, calcein AM, MTT, any fluorescent viability assay<\/td>\n            <td>Albumin, IgG, lipid-binding proteins<\/td>\n            <td>Identical serum % in all wells. Serum-only blank subtraction. <a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">FBS Low Endotoxin<\/a><\/td>\n          <\/tr>\n        <\/tbody>\n      <\/table>\n    <\/div>\n  <\/div>\n\n  <!-- 3 PROTOCOLS -->\n  <div class=\"sec\">\n    <h2>Validated Protocols<\/h2>\n\n    <div class=\"protocol\">\n      <h4>Protocol 1 \u2014 ADCC Assay with FBS Ultra Low IgG<\/h4>\n      <p>1. Expand NK cells or PBMC for 5\u20137 days in 5\u201310% FBS Ultra Low IgG &lt;5 \u00b5g\/mL in RPMI-1640.<br>\n      2. Prepare target cells in 5% FBS Ultra Low IgG \u2014 wash 2\u00d7 before assay to remove residual bovine IgG.<br>\n      3. Opsonise target cells with therapeutic mAb at 1\u201310 \u00b5g\/mL for 30 min at 37\u00b0C.<br>\n      4. Co-culture effector + target at E:T ratio 10:1\u201350:1 in 5% FBS Ultra Low IgG for 4\u20136 hours at 37\u00b0C.<br>\n      5. Read LDH release, calcein AM, or PI flow \u2014 serum concentration identical in spontaneous and maximum lysis controls.<br>\n      <strong>% Specific lysis = [(test \u2212 spontaneous) \/ (maximum \u2212 spontaneous)] \u00d7 100<\/strong><\/p>\n    <\/div>\n\n    <div class=\"protocol\">\n      <h4>Protocol 2 \u2014 CDC Assay with Human Serum AB (native + HI pair)<\/h4>\n      <p>1. Prepare target cells (e.g. Daudi CD20+) in serum-free medium \u2014 wash 2\u00d7.<br>\n      2. Add therapeutic mAb at 1\u201310 \u00b5g\/mL \u2014 incubate 30 min on ice.<br>\n      3. Add Human Serum AB OTC native at 10\u201325% as complement source. Pair with Human Serum AB HI at identical concentration as complement-depleted control.<br>\n      4. Incubate 37\u00b0C, 60 min.<br>\n      5. Read PI exclusion by flow cytometry or LDH release.<br>\n      <strong>Complement-specific lysis = CDC (native) \u2212 CDC (HI)<\/strong><\/p>\n    <\/div>\n\n    <div class=\"protocol\">\n      <h4>Protocol 3 \u2014 AlamarBlue with Consistent Serum Control<\/h4>\n      <p>1. Seed 2,000\u201310,000 cells\/well in 96-well plate in 5\u201310% FBS Low Endotoxin \u22645 EU\/mL.<br>\n      2. Add test compound in serial dilution in medium with identical serum concentration.<br>\n      3. Include: vehicle control, maximum kill control (0.1% Triton X-100), serum-only blank (no cells) \u2014 ALL with identical serum %.<br>\n      4. Add AlamarBlue reagent at 1\/10 volume \u2014 incubate 2\u20134 hours at 37\u00b0C.<br>\n      5. Read fluorescence (Ex 560 \/ Em 590 nm). <strong>Subtract serum-only blank from all wells before calculating % viability.<\/strong><\/p>\n    <\/div>\n  <\/div>\n\n  <!-- FAQ -->\n  <div class=\"faq\">\n    <h2>Frequently Asked Questions<\/h2>\n    <div class=\"faq-item\">\n      <div class=\"faq-q\">Can I use standard FBS for ADCC if I add the right controls?<\/div>\n      <div class=\"faq-a\">No. Controls cannot correct for Fc\u03b3R occupancy by bovine IgG. The interference occurs before the assay reads out \u2014 NK cells pre-loaded with bovine IgG at CD16 have reduced mAb binding capacity. This reduces the measured cytotoxicity signal, not the background. FBS Ultra Low IgG &lt;5 \u00b5g\/mL is the only solution.<\/div>\n    <\/div>\n    <div class=\"faq-item\">\n      <div class=\"faq-q\">Why does CDC require human serum instead of FBS?<\/div>\n      <div class=\"faq-a\">Complement is highly species-specific. Human cells express regulatory proteins (CD46, CD55, CD59) that interact specifically with human complement components. Bovine complement activates through different pathways with different kinetics. CDC data generated with bovine serum does not predict human complement-mediated killing in clinical or translational settings.<\/div>\n    <\/div>\n    <div class=\"faq-item\">\n      <div class=\"faq-q\">What is the correct serum concentration for AlamarBlue?<\/div>\n      <div class=\"faq-a\">The concentration must be identical in every well \u2014 including cell-free blanks, vehicle controls, maximum kill controls, and all compound dilutions. FBS and BSA reduce resazurin signal concentration-dependently. If serum concentration differs between wells, apparent cytotoxicity or protection is generated as a measurement artefact. Subtract the serum-only blank from all wells before calculating % viability.<\/div>\n    <\/div>\n    <div class=\"faq-item\">\n      <div class=\"faq-q\">Which serum for ELISpot with PBMCs from vaccinated donors?<\/div>\n      <div class=\"faq-a\">FBS Very Low Endotoxin \u22641 EU\/mL to eliminate monocyte TLR4 activation. Human Serum AB HI is preferred when a human matrix is needed or complement-mediated PBMC lysis is observed. Heat inactivation is mandatory \u2014 active complement lyses lymphoblasts and activated immune cells during the assay period.<\/div>\n    <\/div>\n    <div class=\"faq-item\">\n      <div class=\"faq-q\">Can NCS or Calf Serum be used for cytotoxicity screening?<\/div>\n      <div class=\"faq-a\">For high-throughput compound screening in standard immortalised cell lines (CHO, HeLa, HEK293, Vero) where IgG levels and complement sensitivity are not critical \u2014 yes. NCS and CS perform equivalently to FBS at lower cost for general MTT\/AlamarBlue assays. For ADCC, CDC, PBMC, or ELISpot assays, use the specific grades listed in this guide.<\/div>\n    <\/div>\n  <\/div>\n\n  <!-- RELATED -->\n  <div class=\"related\">\n    <h3>Related Applications &amp; Products<\/h3>\n    <div class=\"rel-grid\">\n      <a href=\"https:\/\/www.seamlessbio.de\/applications\/pbmc-immunology\">\u2192 PBMC &amp; Immunology Assay Guide<\/a>\n      <a href=\"https:\/\/www.seamlessbio.de\/applications\/car-t-cell-manufacturing\">\u2192 CAR-T Cell Manufacturing Guide<\/a>\n      <a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-ultra-low-igg-low-igg\">\u2192 FBS Ultra Low IgG &lt;5 \u00b5g\/mL<\/a>\n      <a href=\"https:\/\/www.seamlessbio.de\/products\/human-sera-plasma-and-more\/human-serum-off-the-clot-type-ab\">\u2192 Human Serum AB OTC (native)<\/a>\n      <a href=\"https:\/\/www.seamlessbio.de\/products\/fetal-bovine-serum\/fbs-low-very-low-endotoxin\">\u2192 FBS Very Low Endotoxin \u22641 EU\/mL<\/a>\n      <a href=\"https:\/\/www.seamlessbio.de\/products\/albumin-proteins\/bsa-low-endotoxin\">\u2192 BSA Low Endotoxin<\/a>\n    <\/div>\n  <\/div>\n\n  <div class=\"hub-back\"><a href=\"https:\/\/www.seamlessbio.de\/applications\/\">\u2190 All Application Guides Overview<\/a><\/div>\n\n  <div class=\"cta\">\n    <h2>Request Free Test Volumes for Cytotoxicity Assay Validation<\/h2>\n    <p>FBS Ultra Low IgG and Human Serum AB samples available. Lot-specific IgG and endotoxin data per lot. Batch reservation for extended assay campaigns.<br>Email: info@seamlessbio.de | +49 851 37932226<\/p>\n    <div class=\"ctabtns\">\n      <a href=\"mailto:info@seamlessbio.de\" class=\"ctab1\">Contact Us<\/a>\n      <a href=\"https:\/\/shop.seamlessbio.de\" class=\"ctab2\">View in Shop<\/a>\n    <\/div>\n  <\/div>\n<\/div>\n<script type=\"application\/ld+json\">\n{\n  \"@context\": \"https:\/\/schema.org\",\n  \"@graph\": [\n    {\n      \"@type\": \"FAQPage\",\n      \"@id\": \"https:\/\/www.seamlessbio.de\/applications\/cytotoxicity-assays#faqpage\",\n      \"mainEntity\": [\n    {\n      \"@type\": \"Question\",\n      \"name\": \"Can I use standard FBS for ADCC if I add the right controls?\",\n      \"acceptedAnswer\": {\n        \"@type\": \"Answer\",\n        \"text\": \"No. Controls cannot correct for Fc\u03b3R occupancy by bovine IgG. The interference occurs before the assay reads out \u2014 NK cells pre-loaded with bovine IgG at CD16 have reduced mAb binding capacity. This reduces the measured cytotoxicity signal, not the background. FBS Ultra Low IgG &lt;5 \u00b5g\/mL is the only solution.\"\n      }\n    },\n    {\n      \"@type\": \"Question\",\n      \"name\": \"Why does CDC require human serum instead of FBS?\",\n      \"acceptedAnswer\": {\n        \"@type\": \"Answer\",\n        \"text\": \"Complement is highly species-specific. Human cells express regulatory proteins (CD46, CD55, CD59) that interact specifically with human complement components. Bovine complement activates through different pathways with different kinetics. CDC data generated with bovine serum does not predict human complement-mediated killing in clinical or translational settings.\"\n      }\n    },\n    {\n      \"@type\": \"Question\",\n      \"name\": \"What is the correct serum concentration for AlamarBlue?\",\n      \"acceptedAnswer\": {\n        \"@type\": \"Answer\",\n        \"text\": \"The concentration must be identical in every well \u2014 including cell-free blanks, vehicle controls, maximum kill controls, and all compound dilutions. FBS and BSA reduce resazurin signal concentration-dependently. If serum concentration differs between wells, apparent cytotoxicity or protection is generated as a measurement artefact. Subtract the serum-only blank from all wells before calculating % viability.\"\n      }\n    },\n    {\n      \"@type\": \"Question\",\n      \"name\": \"Which serum for ELISpot with PBMCs from vaccinated donors?\",\n      \"acceptedAnswer\": {\n        \"@type\": \"Answer\",\n        \"text\": \"FBS Very Low Endotoxin \u22641 EU\/mL to eliminate monocyte TLR4 activation. Human Serum AB HI is preferred when a human matrix is needed or complement-mediated PBMC lysis is observed. Heat inactivation is mandatory \u2014 active complement lyses lymphoblasts and activated immune cells during the assay period.\"\n      }\n    },\n    {\n      \"@type\": \"Question\",\n      \"name\": \"Can NCS or Calf Serum be used for cytotoxicity screening?\",\n      \"acceptedAnswer\": {\n        \"@type\": \"Answer\",\n        \"text\": \"For high-throughput compound screening in standard immortalised cell lines (CHO, HeLa, HEK293, Vero) where IgG levels and complement sensitivity are not critical \u2014 yes. NCS and CS perform equivalently to FBS at lower cost for general MTT\/AlamarBlue assays. For ADCC, CDC, PBMC, or ELISpot assays, use the specific grades listed in this guide.\"\n      }\n    }\n      ]\n    },\n    {\n      \"@type\": \"BreadcrumbList\",\n      \"@id\": \"https:\/\/www.seamlessbio.de\/applications\/cytotoxicity-assays#breadcrumb\",\n      \"itemListElement\": [\n        {\n          \"@type\": \"ListItem\",\n          \"position\": 1,\n          \"name\": \"Home\",\n          \"item\": \"https:\/\/www.seamlessbio.de\/\"\n        },\n        {\n          \"@type\": \"ListItem\",\n          \"position\": 2,\n          \"name\": \"Applications\",\n          \"item\": \"https:\/\/www.seamlessbio.de\/applications\/\"\n        },\n        {\n          \"@type\": \"ListItem\",\n          \"position\": 3,\n          \"name\": \"Cytotoxicity Assays\",\n          \"item\": \"https:\/\/www.seamlessbio.de\/applications\/cytotoxicity-assays\"\n        }\n      ]\n    }\n  ]\n}\n<\/script>\n<\/section>\t\t\t\t<\/div>\n\t\t\n<\/div>\n\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"<p>Home\/ Applications\/ Cytotoxicity Assays SeamlessBio \u2014 ADCC \u00b7 CDC \u00b7 MTT \u00b7 AlamarBlue \u00b7 LDH \u00b7 ELISpot Serum Selection for Cytotoxicity Assays \u2014 ADCC, CDC, MTT, AlamarBlue, LDH &amp; ELISpot [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_eb_attr":"","footnotes":""},"class_list":["post-1832","page","type-page","status-publish","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.0 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Application Cytotoxicity Assay - SeamlessBio<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"http:\/\/seamlessbio.de\/de\/application-cytotoxicity-assay\/\" \/>\n<meta property=\"og:locale\" content=\"de_DE\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Application Cytotoxicity Assay - SeamlessBio\" \/>\n<meta property=\"og:description\" content=\"Home\/ Applications\/ Cytotoxicity Assays SeamlessBio \u2014 ADCC \u00b7 CDC \u00b7 MTT \u00b7 AlamarBlue \u00b7 LDH \u00b7 ELISpot Serum Selection for Cytotoxicity Assays \u2014 ADCC, CDC, MTT, AlamarBlue, LDH &amp; ELISpot [&hellip;]\" \/>\n<meta property=\"og:url\" content=\"http:\/\/seamlessbio.de\/de\/application-cytotoxicity-assay\/\" \/>\n<meta property=\"og:site_name\" content=\"SeamlessBio\" \/>\n<meta property=\"article:modified_time\" content=\"2026-06-27T11:27:15+00:00\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Gesch\u00e4tzte Lesezeit\" \/>\n\t<meta name=\"twitter:data1\" content=\"7\u00a0Minuten\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"http:\\\/\\\/seamlessbio.de\\\/application-cytotoxicity-assay\\\/\",\"url\":\"http:\\\/\\\/seamlessbio.de\\\/application-cytotoxicity-assay\\\/\",\"name\":\"Application Cytotoxicity Assay - SeamlessBio\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#website\"},\"datePublished\":\"2026-06-27T11:26:18+00:00\",\"dateModified\":\"2026-06-27T11:27:15+00:00\",\"breadcrumb\":{\"@id\":\"http:\\\/\\\/seamlessbio.de\\\/application-cytotoxicity-assay\\\/#breadcrumb\"},\"inLanguage\":\"de\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"http:\\\/\\\/seamlessbio.de\\\/application-cytotoxicity-assay\\\/\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"http:\\\/\\\/seamlessbio.de\\\/application-cytotoxicity-assay\\\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Start\",\"item\":\"https:\\\/\\\/seamlessbio.de\\\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"Application Cytotoxicity Assay\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#website\",\"url\":\"https:\\\/\\\/seamlessbio.de\\\/\",\"name\":\"SeamlessBio\",\"description\":\"Expert for biological materials for pharma, vaccines and research organizations\",\"publisher\":{\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#organization\"},\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\\\/\\\/seamlessbio.de\\\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"de\"},{\"@type\":\"Organization\",\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#organization\",\"name\":\"SeamlessBio\",\"url\":\"https:\\\/\\\/seamlessbio.de\\\/\",\"logo\":{\"@type\":\"ImageObject\",\"inLanguage\":\"de\",\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#\\\/schema\\\/logo\\\/image\\\/\",\"url\":\"https:\\\/\\\/seamlessbio.de\\\/wp-content\\\/uploads\\\/2026\\\/04\\\/cropped-cropped-cropped-\u0438\u0437\u043e\u0431\u0440\u0430\u0436\u0435\u043d\u0438\u0435_2026-04-26_165903047.png\",\"contentUrl\":\"https:\\\/\\\/seamlessbio.de\\\/wp-content\\\/uploads\\\/2026\\\/04\\\/cropped-cropped-cropped-\u0438\u0437\u043e\u0431\u0440\u0430\u0436\u0435\u043d\u0438\u0435_2026-04-26_165903047.png\",\"width\":309,\"height\":88,\"caption\":\"SeamlessBio\"},\"image\":{\"@id\":\"https:\\\/\\\/seamlessbio.de\\\/#\\\/schema\\\/logo\\\/image\\\/\"}}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"Application Cytotoxicity Assay - SeamlessBio","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"http:\/\/seamlessbio.de\/de\/application-cytotoxicity-assay\/","og_locale":"de_DE","og_type":"article","og_title":"Application Cytotoxicity Assay - SeamlessBio","og_description":"Home\/ Applications\/ Cytotoxicity Assays SeamlessBio \u2014 ADCC \u00b7 CDC \u00b7 MTT \u00b7 AlamarBlue \u00b7 LDH \u00b7 ELISpot Serum Selection for Cytotoxicity Assays \u2014 ADCC, CDC, MTT, AlamarBlue, LDH &amp; ELISpot [&hellip;]","og_url":"http:\/\/seamlessbio.de\/de\/application-cytotoxicity-assay\/","og_site_name":"SeamlessBio","article_modified_time":"2026-06-27T11:27:15+00:00","twitter_card":"summary_large_image","twitter_misc":{"Gesch\u00e4tzte Lesezeit":"7\u00a0Minuten"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"WebPage","@id":"http:\/\/seamlessbio.de\/application-cytotoxicity-assay\/","url":"http:\/\/seamlessbio.de\/application-cytotoxicity-assay\/","name":"Application Cytotoxicity Assay - SeamlessBio","isPartOf":{"@id":"https:\/\/seamlessbio.de\/#website"},"datePublished":"2026-06-27T11:26:18+00:00","dateModified":"2026-06-27T11:27:15+00:00","breadcrumb":{"@id":"http:\/\/seamlessbio.de\/application-cytotoxicity-assay\/#breadcrumb"},"inLanguage":"de","potentialAction":[{"@type":"ReadAction","target":["http:\/\/seamlessbio.de\/application-cytotoxicity-assay\/"]}]},{"@type":"BreadcrumbList","@id":"http:\/\/seamlessbio.de\/application-cytotoxicity-assay\/#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Start","item":"https:\/\/seamlessbio.de\/"},{"@type":"ListItem","position":2,"name":"Application Cytotoxicity Assay"}]},{"@type":"WebSite","@id":"https:\/\/seamlessbio.de\/#website","url":"https:\/\/seamlessbio.de\/","name":"SeamlessBio","description":"Experte f\u00fcr biologische Materialien f\u00fcr die Pharmaindustrie, Impfstoffe und Forschungseinrichtungen","publisher":{"@id":"https:\/\/seamlessbio.de\/#organization"},"potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"https:\/\/seamlessbio.de\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"de"},{"@type":"Organization","@id":"https:\/\/seamlessbio.de\/#organization","name":"SeamlessBio","url":"https:\/\/seamlessbio.de\/","logo":{"@type":"ImageObject","inLanguage":"de","@id":"https:\/\/seamlessbio.de\/#\/schema\/logo\/image\/","url":"https:\/\/seamlessbio.de\/wp-content\/uploads\/2026\/04\/cropped-cropped-cropped-\u0438\u0437\u043e\u0431\u0440\u0430\u0436\u0435\u043d\u0438\u0435_2026-04-26_165903047.png","contentUrl":"https:\/\/seamlessbio.de\/wp-content\/uploads\/2026\/04\/cropped-cropped-cropped-\u0438\u0437\u043e\u0431\u0440\u0430\u0436\u0435\u043d\u0438\u0435_2026-04-26_165903047.png","width":309,"height":88,"caption":"SeamlessBio"},"image":{"@id":"https:\/\/seamlessbio.de\/#\/schema\/logo\/image\/"}}]}},"_links":{"self":[{"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/pages\/1832","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/comments?post=1832"}],"version-history":[{"count":3,"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/pages\/1832\/revisions"}],"predecessor-version":[{"id":1835,"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/pages\/1832\/revisions\/1835"}],"wp:attachment":[{"href":"https:\/\/seamlessbio.de\/de\/wp-json\/wp\/v2\/media?parent=1832"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}