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id="layout_20040304" role="draggable" data-appid="panel" data-state="false"> <div class="zbztxd9tjj9" id="layout_20040304_style" class="hide"> </div> <div id="zbztxd9tjj9" class="appMask"></div> <div id="zbztxd9tjj9" class="appContent"> <div id="zbztxd9tjj9" class="phx-panel-wrap" data-showtitle="yes" data-isad="no" data-pattern="fixed" data-place="6" data-width="200" data-height="200"> <div id="zbztxd9tjj9" class="phx-panel-title clearfix"> <span id="zbztxd9tjj9" class="text pull-left">文章詳情</span> </div> <div id="zbztxd9tjj9" class="phx-panel"> <div id="zbztxd9tjj9" class="appWrapper" id="app_24330443" role="draggable" data-type="app" data-appid="datumInfo" data-config="" data-state="true"> <div id="zbztxd9tjj9" class="appMask"></div> <div id="zbztxd9tjj9" class="appContent"> <div id="zbztxd9tjj9" class="datum_info"> <h1 class="title">**細(xì)胞凍存復(fù)蘇技術(shù)</h1> <div id="zbztxd9tjj9" class="bgcolor_01"> <div id="zbztxd9tjj9" class="bgcolor_sum"> <div id="zbztxd9tjj9" class="left">日期：2024-11-22 08:49</div> <div id="zbztxd9tjj9" class="right">瀏覽次數(shù)：552</div> </div> <div id="zbztxd9tjj9" class="bgcolor_sum01"> 摘要： 1. 細(xì)胞凍存復(fù)蘇技術(shù)簡介在ELISPOT檢測的應(yīng)用中，往往需要用到凍存的**細(xì)胞樣品作為檢測細(xì)胞。由于ELISPOT檢測的是細(xì)胞的**功能，不僅僅需要保持細(xì)胞的存活率，而且還要保證細(xì)胞的功能不發(fā)生變化。傳統(tǒng)的細(xì)胞凍存方法通常是為了保存細(xì)胞株，對細(xì)胞存活率的要求不高，更沒考慮保持細(xì)胞原有的**狀態(tài)，所以傳統(tǒng)的細(xì)胞凍存方法已經(jīng)不能滿足ELISPOT檢測的要求。參見圖7.1。考慮到ELISPOT檢測的特殊性，荷蘭U-CyTech公司經(jīng)過精心的研究與反復(fù)的驗證，推出了Cryostar?凍存試劑盒，在凍存液中添加了一系列對細(xì)胞有特殊保... </div> </div> <div id="zbztxd9tjj9" class="content"> <h4 style="margin:0px 0px 0px 42pt;padding:0px;font-weight:normal;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:18px;font-family:微軟雅黑;color:#333333;">1. </span><span style="font-size:18px;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存復(fù)蘇技術(shù)簡介</span><span style="font-size:12pt;font-family:微軟雅黑;"></span> </h4> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">在ELISPOT檢測的應(yīng)用中，往往需要用到凍存的**細(xì)胞樣品作為檢測細(xì)胞。由于ELISPOT檢測的是細(xì)胞的**功能，不僅僅需要保持細(xì)胞的存活率，而且還要保證細(xì)胞的</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">功能不發(fā)生變化。傳統(tǒng)的細(xì)胞凍存方法通常是為了保存細(xì)胞</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">株</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，對細(xì)胞存活率的要求不高，更沒考慮保持細(xì)胞原有的**狀態(tài)，所以傳統(tǒng)的細(xì)胞凍存方法已經(jīng)不能滿足ELISPOT檢測的要求。</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">參見圖7.1。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">考慮到ELISPOT檢測的特殊性，荷蘭</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">U</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">-CyTech公司經(jīng)過精心的研究與反復(fù)的驗證，推出</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">了</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Cryostar?凍存試劑盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，在凍存液中添加了一系列對細(xì)胞有特殊保護(hù)作用的保護(hù)劑。達(dá)科為公司在接到Cryostar?凍存試劑盒之后，在我們的實(shí)驗室以及國內(nèi)數(shù)家客戶的實(shí)驗室做了細(xì)胞凍存與復(fù)蘇的實(shí)驗，并且對復(fù)蘇之后的細(xì)胞做了ELISPOT檢測。結(jié)果表明，**細(xì)胞的存活率超過90%，ELISPOT斑點(diǎn)形成頻率與新鮮細(xì)胞完全相同。</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">參見圖</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">6</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">.1。</span><span style="font-size:9pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-align:center;text-indent:21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"><img src="http://www.bio-city.net/uploads/common/image/20130829/20130829-170227_713793528.jpg" width="600" alt="" style="border-style:none;vertical-align:top;display:block;margin:0px auto;"><br> </span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2005年底，在達(dá)科為公司與中國生物制品檢定所合作制備凍存的質(zhì)控細(xì)胞庫時，</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar?</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2005年底，在達(dá)科為公司與中國生物制品檢定所合作制備凍存的質(zhì)控細(xì)胞庫時，</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar?凍存試劑盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">與本文的凍存復(fù)蘇技術(shù)經(jīng)受住了嚴(yán)格的考驗，細(xì)胞存活率達(dá)到95%。這表明，該項技術(shù)已經(jīng)完全成熟，可以用于建立大型凍存細(xì)胞庫（10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">8</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">-10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">9</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">cells）。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">凍存試劑盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">與本文的凍存復(fù)蘇技術(shù)經(jīng)受住了嚴(yán)格的考驗，細(xì)胞存活率達(dá)到95%。這表明，該項技術(shù)已經(jīng)完全成熟，可以用于建立大型凍存細(xì)胞庫（10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">8</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">-10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">9</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">cells）。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:15.75pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">根據(jù)我們的實(shí)驗經(jīng)驗，**的細(xì)胞凍存效果需要上等的凍存試劑</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">加上嚴(yán)格規(guī)范的實(shí)驗操作。二者缺一不可，不可偏頗</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，千萬不要因為有了好的試劑盒而放松對實(shí)驗操作的要求。</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">為了幫助國內(nèi)的客戶解決好細(xì)胞凍存的問題，達(dá)科為公司以</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar? 凍存試劑盒使用說明為例</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">特此編撰這份詳細(xì)的細(xì)胞凍存、復(fù)蘇實(shí)驗操作手冊，僅供參考。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:15.75pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryosta</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">r?</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">凍存試劑盒（貨號U</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">H-F1002-050</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">）的內(nèi)容如下：</span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar? 重懸液(Cryostar? Res</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">u</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">spension Medium)，1瓶，液體，25m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l。</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">-20 °C</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">長期</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">保存</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，一旦開始使用，放置于4</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">°C</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">冰箱。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar? 2</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">×</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">凍存液(Cryostar? 2</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">×</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> Freezing Medium)，1瓶，液體，25m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，-20 °C</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">長期</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">保存</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，一旦開始使用，放置于4</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">°C</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">冰箱。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">英文原裝操作手冊一本；中文詳細(xì)操作手冊一本。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <h4 style="margin:0px 0px 0px 42pt;padding:0px;font-weight:normal;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:18px;font-family:微軟雅黑;color:#333333;">2. </span><span style="font-size:18px;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存、復(fù)蘇的標(biāo)準(zhǔn)程序</span><span style="font-size:14pt;font-family:微軟雅黑;"></span> </h4> <p style="margin-top:0px;margin-bottom:0px;margin-left:31.5pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:Wingdings;color:#333333;">l </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">實(shí)驗儀器：</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">可控溫度細(xì)胞離心機(jī)離心機(jī)</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">；</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">血球計數(shù)板或者自動</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">細(xì)胞計數(shù)設(shè)備； </span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">細(xì)胞</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">程序降溫</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Nalgene </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">“Mr. Frosty”；</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">-70°C冰箱；</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">液氮罐；</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">37°C水浴鍋。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:31.5pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:Wingdings;color:#333333;">l </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">耗材</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">準(zhǔn)備</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">15m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">圓錐底聚丙烯</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（PP）</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">離心管</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">； </span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">無菌的細(xì)胞凍存管</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，外螺旋；</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:31.5pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:Wingdings;color:#333333;">l </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">試劑</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">配制</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">熱失活</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">補(bǔ)體</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">胎牛血清</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">；</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> </span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">R0培養(yǎng)基：RPMI-1640培養(yǎng)基，含谷氨酰胺，加入1%雙抗，不加血清。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">達(dá)優(yōu)®無血清培養(yǎng)基：已經(jīng)含有谷氨酰胺和雙抗，打開即用。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar?重懸液(Cryostar? Res</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">u</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">spension Medium)：含有細(xì)胞凍存保護(hù)成分，-20°C長期保存，4°C可以保存6個月。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">Cryostar? 2</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">×</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">凍存液</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（Cryostar? Freezing Medium）：含有細(xì)胞凍存保護(hù)成分以及DMSO，-20°C長期保存，4°C可以保存6個月。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:-3.57pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21.45pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:Wingdings;color:#333333;"> 2 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">臺盼藍(lán)</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:31.5pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:Wingdings;color:#333333;">l </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存程序</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">1. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">將備用</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存管</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">做上詳細(xì)記號。</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存管</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">、</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">細(xì)胞凍存盒</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">、</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Cryostar? 重懸液</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">、</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Cryostar? 2</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">×</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">凍存液</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">放入4 </span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">對要凍存細(xì)胞進(jìn)行計數(shù)，然后4</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;"> o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C，400g離心10 min。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">3. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（冰上操作）棄上清，根據(jù)細(xì)胞計數(shù)結(jié)果加入4</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;"> o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C的</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Cryostar? 重懸液</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">重懸，使細(xì)胞濃度調(diào)整到兩倍的凍存濃度（比如想要凍存的細(xì)胞濃度為：1×10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">7</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> cells/m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，就用重懸液將細(xì)胞濃度調(diào)整到2×10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">7</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">/m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">），然后將細(xì)胞吹打均勻。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:24pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">注意：</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">重懸液不含有DMSO，所以這一步動作可以稍微劇烈。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">4. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（冰上操作）然后</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">逐滴加入</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">等體積冰浴的</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Cryostar? 2</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">×</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">凍存液</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，（細(xì)胞濃度變?yōu)?×10</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">7 </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">cells/m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">）。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">注意：</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">一定要逐滴加入，避免溶液中DMSO濃度的劇烈變化，詳見后面的說明。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">5. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（冰上操作）以每只1m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">細(xì)胞懸液的量分裝入在-20</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡過的凍存管，然后將凍存管的蓋子擰嚴(yán)實(shí)。否則液氮會滲漏進(jìn)來。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">6. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">立即將凍存管放入已在4</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡的凍存盒(“Mr. Frosty”)，然后將凍存盒放入-70</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C冰箱內(nèi)。也可以將凍存管放于程序降溫儀中，以1</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C/min的速率降溫到–70</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">7. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">24</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">hr</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">后，把-70</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C保存的凍存管轉(zhuǎn)移到液氮中。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">8. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">將細(xì)胞在液氮罐中凍存位置記錄在實(shí)驗室的液氮設(shè)備管理單上。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:31.5pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-21pt;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:Wingdings;color:#333333;">l </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">細(xì)胞復(fù)蘇程序</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">1.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">血清、</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">R0培養(yǎng)基需要在4</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡，</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Lympho-Spot?無血清培養(yǎng)基</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">需要37</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡。</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">15</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">離心管需要4</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C熱平衡。水浴鍋要預(yù)先調(diào)到37</span><span style="font-size:10.5pt;font-family:微軟雅黑;vertical-align:super;color:#333333;">o</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">C。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">用夾子將細(xì)胞凍存管從液氮中取出。如果有液氮滲漏到凍存管中，要稍微擰開管蓋，讓氣化的液氮逃逸。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">警告：據(jù)報道，有些凍存管在解凍時會發(fā)生爆炸，為了消除這種危險，建議實(shí)驗中只使用牢固的聚乙烯凍存管用于液氮保存。</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">3.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">用夾子持住凍存管，浸入37oC水浴鍋內(nèi)。輕輕晃動凍存管，注意管內(nèi)凍存細(xì)胞的融化情況。當(dāng)管內(nèi)的</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">冰核還有黃豆大小</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">的時候，取出來，轉(zhuǎn)移到超凈工作臺內(nèi)。用酒精棉擦拭管口，然后將凍存管插于碎冰之上。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">注意：這一步對細(xì)胞存活率至關(guān)重要。既要盡快融化，以減少融化過程對細(xì)胞的損害；又要盡可能減少細(xì)胞在較高溫度停留的時間，以減少DMSO對細(xì)胞的毒害。切不可將凍存管放在在燒杯內(nèi)不管。</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">4.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（冰上操作）</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">用移液槍取1ml胎牛血清</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">(4oC)</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，槍頭插到凍存管</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">管底</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">緩慢地</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">加入這一毫升血清。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">5.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（冰上操作）</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">溫柔</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">地</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">將凍存管內(nèi)的細(xì)胞懸液吸取出來，轉(zhuǎn)移到一個預(yù)冷的</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">15</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">離心管內(nèi)。然后，</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">用移液槍取1mlR0培養(yǎng)基</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">(4oC)</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，槍頭插到離心管</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">管底</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">緩慢地</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">加入這一毫升培養(yǎng)基。然后換槍頭再重復(fù)兩次，一共向離心管管底加入3ml的R0培養(yǎng)基。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">6. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">蓋上離心管管蓋，輕輕上下</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">顛倒混勻</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">。然后緩慢的補(bǔ)加R0培養(yǎng)基到14ml，此時不再需要從管底加入了。補(bǔ)加滿，然后蓋上管蓋，輕輕的上下</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">顛倒混勻</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">7. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">4oC，400g離心10min，棄上清，加入1m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> </span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Lympho-Spot?無血清培養(yǎng)基</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">，混合均勻。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">8.</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">（優(yōu)化步驟，可以略過）如果細(xì)胞有成團(tuán)現(xiàn)象，這是由于死亡細(xì)胞釋放出來的DNA纏繞所致?？梢约尤?.1m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> DNase(1mg/m</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">)，37 oC孵育10min。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;margin-left:27pt;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;text-indent:-18pt;text-align:justify;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">9. </span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">取</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">2</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">0μ</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">l</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">細(xì)胞懸液，臺盼藍(lán)活細(xì)胞計數(shù)，計算存活率（viability）。</span><span style="font-size:10.5pt;font-family:微軟雅黑;"></span> </p> <p style="margin-top:0px;margin-bottom:0px;padding:0px;font-family:'Microsoft Yahei', arial, 宋體;line-height:21.6px;white-space:normal;background-color:#FFFFFF;"> <span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;"> 根據(jù)計數(shù)結(jié)果，補(bǔ)加</span><span style="font-size:10.5pt;font-weight:bold;font-family:微軟雅黑;color:#333333;">Lympho-Spot?無血清培養(yǎng)基</span><span style="font-size:10.5pt;font-family:微軟雅黑;color:#333333;">至所需要的細(xì)胞濃度，進(jìn)行ELISPOT檢測。</span> </p> </div> <div id="zbztxd9tjj9" class="datumrelate"> <span>下一篇：</span> <a href="/klbscience_Article_7246497.html" title="內(nèi)**對ELISPOT檢測的干擾">內(nèi)**對ELISPOT檢測的干擾</a> <br> <span>上一篇：</span> <a href="/klbscience_Article_7385113.html" title="ELISPOT技術(shù)的歷史與發(fā)展">ELISPOT技術(shù)的歷史與發(fā)展</a> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <footer id="footer" class="footer" data-appid="layout"> <div class="zbztxd9tjj9" id="footerWrapper" class="footerWrapper" data-appid="wrapper"> <div class="zbztxd9tjj9" id="footerContainer" class="footerContainer" data-appid="container"> <div class="zbztxd9tjj9" id="app_55124555_style" class="hide"> </div> <div 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