产品描述
Calcein, AM是一种可对活细胞进行荧光标记的细胞染色试剂，它穿透细胞膜进入细胞后被细胞内的酯酶剪切形成Calcein，从而被滞留在细胞内，发出强绿色荧光。与其它同类试剂（如BCECF, AM和CFDA）相比，Calcein, AM的细胞毒性很低。Calcein的激发和发射波长分别为490 nm和515 nm。
Calcein, AM仅对活细胞染色。作为核染色染料的PI不能穿过活细胞的细胞膜，它穿过死细胞膜的无序区域而到达细胞核，并嵌入细胞的DNA双螺旋从而产生红色荧光（激发：535 nm，发射：617 nm），因此PI仅对死细胞染色。由于Calcein和PI-DNA都可被490 nm激发，因此可用荧光显微镜同时观察活细胞和死细胞。用545 nm激发，仅可观察到死细胞。根据以上特点，Calcein, AM和PI经常被结合用来作为活细胞和死细胞的双重染色。
由于不同细胞系的最佳染色条件不同，我们建议个别确定Calcein, AM和PI的合适浓度。
使用方法
（1）用DMSO制备1 mM 的Calcein, AM溶液，并用PBS将其稀释制成1～50 μM的Calcein, AM溶液。
（2）将1/10细胞培养基体积的Calcein, AM溶液加入到细胞培养基中。
（3）在37℃培养细胞15～30分钟。
（4）用PBS或适当的缓冲液洗涤细胞两次。
（5）用490 nm激发波长，515 nm发射波长的滤光片的荧光显微镜观察细胞。
a) 如果Calcein, AM很难进入细胞，可以使用表面活性剂，如Pluronic F127。
b) 也可以用1/10浓度的Calcein, AM溶液代替培养基。
Calcein, AM is a cell staining reagent that can fluorescently label living cells. After it penetrates the cell membrane and enters the cell, it is cleaved by the intracellular esterase to form Calcein, which is retained in the cell and emits strong green fluorescence. Compared with other similar reagents (such as BCECF, AM and CFDA), Calcein, AM has very low cytotoxicity. The excitation and emission wavelengths of Calcein are 490 nm and 515 nm, respectively.
Calcein, AM only stain live cells. PI, which is a nuclear staining dye, cannot pass through the cell membrane of living cells. It passes through the disordered area of the dead cell membrane to reach the nucleus, and is embedded in the cell’s DNA double helix to generate red fluorescence (excitation: 535 nm, emission: 617 nm). PI only stains dead cells. Since both Calcein and PI-DNA can be excited at 490 nm, a fluorescence microscope can be used to observe live and dead cells simultaneously. With 545 nm excitation, only dead cells can be observed. Based on the above characteristics, Calcein, AM and PI are often combined to double stain live and dead cells.
Due to the different optimal staining conditions for different cell lines, we recommend determining the appropriate concentrations of Calcein, AM and PI individually.
Instructions
(1) Prepare 1 mM Calcein, AM solution with DMSO, and dilute it with PBS to make 1-50 μM Calcein, AM solution.
(2) Add 1/10 of the volume of the cell culture medium Calcein, AM solution to the cell culture medium. b)
(3) Incubate the cells at 37°C for 15-30 minutes.
(4) Wash the cells twice with PBS or an appropriate buffer.
(5) Observe the cells with a fluorescence microscope with a filter with excitation wavelength of 490 nm and emission wavelength of 515 nm.
a) If it is difficult for Calcein and AM to enter cells, you can use surfactants such as Pluronic F127.
b) It is also possible to use 1/10 concentration of Calcein, AM solution instead of medium.
L2518650
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