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  15842062061 2019-04-19 17:19:11

  <p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">細(xì)胞計(jì)數(shù)和存活測(cè)試</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">1. 原理：</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">1.1. 計(jì)算細(xì)胞數(shù)目可用血球計(jì)數(shù)盤(pán)或是Coulter counter 粒子計(jì)數(shù)器自動(dòng)計(jì)數(shù)。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">1.2. 血球計(jì)數(shù)盤(pán)一般有二個(gè)chambers，每個(gè)chamber 中細(xì)刻9 個(gè)1 mm2 大正方形，其中4 個(gè)角落之正方形再細(xì)刻16 個(gè)小格，深度均為0.1 mm。當(dāng)chamber 上方蓋上蓋玻片后，每個(gè)大正方形之體積為1 mm2 x 0.1 mm=1.0 x 10-4 ml。使用時(shí)，計(jì)數(shù)每個(gè)大正方形內(nèi)之細(xì)胞數(shù)目，乘以稀釋倍數(shù)，再乘以104，即為每ml 中之細(xì)胞數(shù)目。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">1.3. 存活測(cè)試之步驟為dye exclusion，利用染料會(huì)滲入死細(xì)胞中而呈色，而活細(xì)胞因細(xì)胞膜完整，染料無(wú)法滲入而不會(huì)呈色。一般使用藍(lán)色之trypan blue 染料，如果細(xì)胞不易吸收trypan blue，則用紅色之Erythrosin bluish。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2. 材料：</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.1. 0.4 % w/v trypan blue (GibcoBRL 15250-061)</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.2. Erythosin bluish stain</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">取0.1 gram Erythrosin bluish (Sigma E-9259) 及0.05 gram preservative methyl</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">paraben (Sigma H-3647) 溶于100 ml Ca++/Mg++ free saline</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.3. 血球計(jì)數(shù)盤(pán)及蓋玻片(Hemocytometer and coverslip)</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.4. 計(jì)數(shù)器(counter)</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.5. 低倍倒立顯微鏡</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">2.6. 粒子計(jì)數(shù)器(Coulter counter, Coulter Electronics)</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">3. 步驟：</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">3.1. 取50ml 細(xì)胞懸浮液與50ml trypan blue (or Erythrosin bluish) 等體積混合均勻于1.5ml 小離心管中。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">3.2. 取少許混合液(約15ml) 自血球計(jì)數(shù)盤(pán)chamber 上方凹槽加入，蓋上蓋玻片，于100 倍倒立顯微鏡下觀察，活細(xì)胞不染色，死細(xì)胞則為藍(lán)色(或紅色- Erythrosin bluish)。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">3.3. 計(jì)數(shù)四個(gè)大方格之細(xì)胞總數(shù)，再除4，乘以稀釋倍數(shù)(至少乘以2，因與trypan blue 等體積混合)，Z后乘以104 ，即為每ml 中細(xì)胞懸浮液之細(xì)胞數(shù)。若細(xì)胞位于線(xiàn)上，只計(jì)上線(xiàn)與右線(xiàn)之細(xì)胞(或計(jì)下線(xiàn)與左線(xiàn)之細(xì)胞)。</p><p style=" font-family:&#39;Microsoft YaHei&#39;;font-size:16px;line-height: 2em;position: relative;">4 大格*細(xì)胞總數(shù)x 2 x 104 / 4= 細(xì)胞數(shù)/ml</p><p><br/></p>

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