Calcein-AM/PI细胞双染试剂盒货号:C542

Calcein-AM/PI细胞双染试剂盒货号:C542
活死细胞双染试剂盒
Calcein-AM/PI Double Staining Kit
商品信息
储存条件:-20度保存
运输条件:室温

特点:

● 经典细胞活死双染荧光检测试剂盒

● EX488 nm同时检测活死细胞

选择数量:

选择规格:
500 tests
3000 tests
价格:
¥1640.00现货(会员请登录查看专享价!)
细胞染色
活动进行中
试剂盒内含
产品概述
荧光特性
染色例
操作步骤
注意事项
参考文献

活动进行中

订购满5000元,200元礼品等你拿

凑单关联产品TOP5

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NO.2.    Annexin V, FITC Apoptosis Detection Kit    细胞凋亡检测

NO.3.    FerroOrange    细胞亚铁离子检测

NO.4.    Liperfluo    细胞脂质过氧化物检测

NO.5.    ROS Assay Kit    活性氧检测

试剂盒内含

1628751749789080.png

产品概述

Calcein-AM/PI细胞双染试剂盒内含两种染料:Calcein-AM和Propidium Iodide (PI)。这个试剂盒可在荧光显微镜下同时观察在同一个细胞培养皿中的活细胞和死细胞。Calcein-AM可透过细胞膜,通过活细胞内的酯酶作用脱去AM基团,产生的Calcein (钙黄绿素) 发出强绿色荧光,因此活细胞在荧光显微镜下可被检测到绿色荧光。另一方面PI可以通过受损的细胞膜进入到死细胞内并嵌入细胞的DNA双螺旋从而产生红色荧光,因此死细胞会被检测到红色荧光。除了用荧光显微镜外,也有报道可以用流式细胞仪和荧光酶标仪来进行定量检测。

荧光特性

Calcein-AM : λex=490 nm , λem=515 nm

PI : λex=530 nm , λem=580 nm

染色例

                                                                           细胞染色实例

1622168981542821.jpg

(a)                                     (b)                                      (c)

a)  Calcein-AM染FTC细胞(活细胞单染)

b)  PI染HCT116细胞(死细胞单染)

c)  Calcein-AM、PI染MHD-1 细胞(活死细胞双染)

最佳浓度摸索

由于不同细胞种类、细胞浓度的染色条件不同,我们建议自行摸索一下Calcein-AM和PI的最适浓度。

Calcein-AM和PI的最佳浓度是根据不同的细胞种类而定,通过以下的操作,我们可以找到不同细胞染色试剂的最佳浓度:

1. 通过在0.1%皂苷或0.1-0.5%毛地黄皂苷中培养10 min或通过在70%乙醇中培养30 min制备死细胞。

2. 用0.1-10 μM PI溶液染死细胞,以便找到仅针对细胞核染色而不对细胞质染色的PI浓度。

3. 用0.1-10 μM Calcein-AM溶液染死细胞,以便找到不对细胞质染色的Calcein-AM浓度,再以此浓度的Calcein-AM对活细胞染色以检验活细胞是否被染色。

操作步骤

以HeLa细胞为例

制备1 mmol/l的Calcein-AM储存液

【500 次】

将200 μl DMSO加入到含200 μg Calcein-AM粉末的管中,用移液器吹打溶解。

【3000 次】

将1 ml DMSO加入到含1 mg Calcein-AM粉末的管中,用移液器吹打溶解。

※Calcein-AM储存液需要避光,在-20℃密封保存。

 

制备染色工作液

将Calcein-AM储存液和PI储存液恢复至室温后使用。

在5 ml的PBS(-)中加入10 μl Calcein-AM储存液和15 μl PI储存液,混匀制成工作液。此时Calcein-AM的浓度为2 μmol/l,而PI的浓度为4.5 μmol/l。

 

染色步骤

1、 用Trypsin-EDTA消化细胞。

2、 通过离心收集细胞(1,000 rpm,3 min)。

3、 去除上清液,加入PBS(-)制备细胞悬液(105 – 106 cells/ml为宜)。

4、 重复步骤2和步骤3数次以消除培养基中的酯酶活性。

5、 取100 μl 染色工作液与200 μl 细胞悬液混合,在37℃培养15 min。

6、 在490±10 nm激发波长下同时观察黄绿色荧光的活细胞和红色荧光的死细胞。另外用545 nm激发波长单独观察死细胞。

注意事项

1、 由于本试剂盒中的Calcein-AM Reagent 粉末和PI Stock Solution量很少,有可能会粘在盖子或管壁上,开封前请先涡旋以使其振落下来。

2、 由于Calcein-AM储存液对潮气敏感,请在使用后密闭Calcein-AM储存液的盖子。如果不能一次用完,建议分装保存,例如分装成10 μl/管,用封口膜封口,并用铝箔纸包裹,放在一个密闭性能好的塑料袋中,并放入一包干燥剂,在≤-20℃密封避光保存。

3、 配制好的染色工作液请在当天使用。

4、 PI有疑似致癌性,使用前应注意以下几点:

1) 使用时请带好手套,口罩,防护眼镜等,不要接触到或呼吸到。

2) 当PI不慎接触到皮肤时,请立刻用大量的水冲洗。

3) 处理方法

清洗容器的清洗液和废液请按照实验室的有毒有害物质的处理方法进行处理,或按照以下方法处理:

・用UV照射的方法进行分解

・用次氯酸钠氧化分解后,进行中和处理

参考文献

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Materials Today, 2020, doi.org/10.1016/j.mattod.2019.12.005

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Advanced Science, 2018, 5, 1800049

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Advanced Functional Materials, 2019, 29(51), 1906569

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Advanced Functional Materials, 2018, 28(35), 1802833

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ACS Nano, 2019, 13(4), 4379-4391

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ACS Nano, 2016, 10(1), 984-97

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