产品描述

台盼蓝(Trypan Blue)，一种偶氮、亲水性酸性蓝色染料，可透过死亡细胞和垂死细胞的细胞膜，将其染成蓝色，而活 细胞由于其细胞膜的完整性， 可将台盼蓝排斥在外， 因此， 通过颜色的变化即可将活细胞(活细胞呈透明无色) 和死细胞鉴 别开来， 基于此原理， 本品广泛用于细胞活性水平的常规评估。台盼蓝还可染色胶原和淀粉样蛋白。台盼蓝与蛋白结合形成 的复合物可发出红色荧光。另外，台盼蓝(合适浓度)还常用来淬灭细胞表面的自荧光或者其他荧光信号。

本试剂盒各组分均以无菌形式提供， 可直接用于细胞实验。按照单次使用量 0.1mL，40208ES60 和 40208ES76 分别可供 检测 100 次和 500 次。

产品组分

运输和保存方法

室温运输和保存即可，二年有效。

注意事项

1) 细胞计数前需对细胞进行充分的清洗， 因为细胞对血清蛋白具有非常高的亲和力， 残留血清蛋白会导致较暗的染色背景。 2)台盼蓝对人体有害，请注意小心防护。

3)为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用方法

1)对于悬浮细胞，离心收集细胞，对其充分清洗后，加入适量细胞重悬液重悬细胞制成单细胞悬液；对于贴壁细胞，先用 胰酶消化细胞，再按照悬浮细胞的方法制备单细胞悬液。

2)取 0.1 mL 单细胞悬液，按照 1:1 比例与 0.1 mL 0.4%台盼蓝染色液充分混匀，室温染色 3~10 min。

 3) 取少量上述染色细胞加入血细胞计数板， 于显微镜低倍镜下计数， 分别计算着色细胞(即损伤细胞和死细胞) 和总细胞。 【注 1】：用移液枪加染色细胞时，沿着盖玻片的边缘轻轻加液使其完全覆盖住整个小室。不可过量加液或者不足量。   【注 2】： 1 mm2 方格内细胞数通常控制在 20-50 cells，当细胞数超过 200 个，需重新调整稀释倍数。

4)按照以下公式来计算细胞数目和活细胞百分比，

a，计算每 mL 细胞数目： Cells/mL=1 mm2 大方格内的平均细胞数×稀释倍数×104；

【注】：此时的稀释倍数为：步骤 2 所取的单细胞悬液与台盼蓝染色液混合后的稀释倍数

b ，总细胞数目： Total cells=  (Cells/mL) ×最初制备单细胞悬液的总体积

c，细胞活力值： Viability  (%) =  (总细胞数-总着色细胞数) /总细胞数×100

【注】：通常情况，健康的对数期培养细胞，活细胞至少占到 95%。

HB220505

苏木素（Hematoxylin）是一种从洋苏木提取的天然染料，以Mayer’s，Gill’s和Harris等多种配方形式普遍用于细胞核，线粒体，粘蛋白，弹性纤维，肌肉，胶原，髓鞘和磷脂的染色，检测原理是带正电荷的碱性染料苏木素与带负电荷的酸性物质结合使其被染成蓝色。

伊红（Eosin），又称曙红，一种红色荧光染料，在水中解离成带负电荷的阴离子，能够与蛋白质氨基中带正电荷的阳离子结合从而使细胞质、红细胞、胶原、肌纤维、结缔组织、嗜伊红颗粒等染成不同程度的红色或者粉红色。作为一种酸性染料，常用于苏木素-伊红染色法（H&E Staining），用作苏木素的复染剂。

苏木素-伊红（H&E）的结合染色使得细胞质呈红色，细胞核显蓝紫色，红细胞呈桔红色，其它成分呈深浅不同的红色，可用于免疫组化中组织切片，血涂片，骨髓切片的染色，也能用于细胞涂片的染色，是细胞生物学、组织学及病理学等学科必不可少的最常规染色方法之一。

产品组分

运输和保存方法

室温运输；室温保存，有效期至少1年。

注意事项

1）为了您的安全和健康，请穿实验服并戴一次性手套操作。
2）本产品仅作科研用途！

使用方法

一、样本前处理

1）石蜡切片

①脱蜡：用无毒环保脱蜡剂或二甲苯脱蜡，10-15 min/次，共2缸2次；

②梯度入水：95%、70%、30%乙醇各2 min，温水2 min，如果脱蜡不干净，需再次温水2 min。此时玻片上除样本部分略有水分外，玻片其余部分均应无水珠。

2）冰冻切片

①回温：按以下方法将预先制作好的并保存在-20℃的冰冻切片取出回温（选取其中一种即可）：

A、室温放置回温5-10 min。

B、37℃孵育箱中进行回温。

C、在37℃水浴箱中放置一个小盒子，再将从-20℃取出的冰冻切片放到小盒当中，目的是让冰冻切片回温至37℃。

②水合：将回温好的切片，水中浸泡30-60 s左右。

【注】：冰冻切片最好用防脱载玻片，切好的片子如不及时染色可放-20℃保存，染色前最好不要用乙醇等固定，否则易造成掉片。

3）血涂片及骨髓涂片

①推片：取全血3 µL左右置载玻片上，将推玻片与载玻片保持30°角，置于血滴正前方，稍往后移与血滴接触，血滴沿推片下缘散开，再匀速沿载玻片平面平稳向前滑动，至血液铺完血膜为止。

②涂片空气中自然干燥，95%乙醇固定2-3 min，水洗约30-60 s。
 

二、 样本染色

1）组织润湿：染色前用蒸馏水润湿组织1-2 min，确保蒸馏水覆盖整个组织，使水分均匀分布；

2）胞核染色：苏木素染色液染色5 min，水洗3-5 s。

3）胞浆染色：可选用以下方法中其中一种进行染色：

①伊红染色液染色10-30 s，用增色液冲洗1-2次，滤纸吸干或自然晾干；

【注】：此法可立即封片镜检。

②伊红染色10-30 s，在水中蘸1-2次（10 s左右），滤纸吸干或自然晾干；

【注】：此法需无水乙醇脱水二次后再封片镜检。

③伊红染色2 min，用蒸馏水洗1-2 min，滤纸吸干或自然晾干；

【注】：此法需无水乙醇脱水二次后再封片镜检。

三、 镜检结果

胞浆呈红色，胞浆呈蓝紫色，红细胞呈桔红色，其它成分呈深浅不同红色。

【注】：若染色较浅，可延长染色时间或者重复染色一次；若染色较深，可延长水冲时间或者再重复梯度脱水一次。

相关产品

      HB190108

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