Calcein AM /PI试剂盒

产品介绍

钙黄绿素-AM (Calcein-AM) 和碘化丙啶 (PI) 溶液，分别对活细胞和死细胞染色，可用于同时对活细胞和死细胞进行荧光染色。Calcein-AM的乙酸甲基酯亲脂性很高，使其可透过细胞膜。尽管Calcein-AM本身并不是荧光分子，但通过活细胞内的酯酶作用，Calcein-AM能脱去AM基，产生的Calcein能发出强绿色荧光 (激发: 490 nm，发射: 515 nm)。因此Calcein-AM仅对活细胞染色。另一方面，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)。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。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。

I. 试剂  Calcein-AM 2mM 50uL in DMSO;  PI (1.5mM) 150uL in water

II. 用荧光显微镜观察细胞形态

以HeLa细胞染色为例，请注意不同的细胞种类、不同浓度，有不同的观察条件。根据细胞条件，摸索不同条件下的细胞贴壁情况和试剂浓度的配制等最佳条件。

1. 染色溶液的配制

1) 用1 ml无水DMSO溶解2 mg Calcein-AM，制备成2 mmol/l的Calcein-AM储备液，-20℃下密闭冷冻保存。

2) 用1 ml ddH2O溶解2 mg PI，制备成3 mmol/l的PI储备液，-20度下密闭冷冻保存，可以保存一年。

3) 将Calcein-AM储备液和PI储备液放置于室温。

4) 加5 µl Calcein-AM储备液和15 µl PI储备液至5 ml PBS中配制成染色溶液。Calcein-AM的终浓度为2 µmol/l，PI的终浓度为4.5 µmol/l。

2. 细胞染色

1) 染色HeLa细胞等贴壁细胞时，先用Trypsin-EDTA等消化细胞，制备成细胞悬液。

2) 将细胞悬液离心3分钟 (1,000 rpm)。

3) 去除上清液，加入PBS缓冲液，细胞数量调整至10e5-10e6个/ml。再用移液器充分混匀。

4) 由于培养基中的血清等含有酯酶，Calcein-AM遇水会分解，会导致空白上升，所以需要离心数次，用PBS洗涤数次直到完全洗净。

5) 将200 µl细胞悬液移至小试管中，加入100 µl染色溶液，在37℃下孵育15分钟。

6) 在盖玻片上滴加适量的染色的细胞溶液。

7) 在荧光显微镜下，先用490±10 nm波长激发，观察黄绿色的活细胞，还可以同时观察到红色的死细胞，然后用545 nm波长激发，能够看到红色的死细胞。

3. 染色试剂的最佳浓度

Calcein-AM和PI最佳浓度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%毛地黄皂苷中孵育10分钟或通过在70%乙醇中孵育30分钟制备死细胞。

2) 用0.1-10 µM PI溶液对死细胞染色，以便找到仅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溶液对死细胞染色，以便找到不对细胞质染色的Calcein-AM浓度。接着用该浓度的Calcein-AM对活细胞染色以检验活细胞可否被染色。

III. 注意事项

1) Calcein-AM的ester部位遇到湿气会分解，使用后请在-20度下密闭冷冻保存，防止水分进入。Calcein-AM储备液用缓冲液或培养基等稀释时尽量现配现用。

2) 使用时一定要带手套、眼罩、口罩。万一接触到皮肤的话，迅速使用大量水清洗。

Product description

Calcein-AM (Calcein-AM) and propidium iodide (PI) solutions stain live and dead cells respectively, and can be used to stain live and dead cells simultaneously. The methyl acetate of Calcein-AM is highly lipophilic, allowing it to penetrate cell membranes. Although Calcein-AM itself is not a fluorescent molecule, through the action of esterase in living cells, Calcein-AM can remove the AM group, and the generated Calcein can emit strong green fluorescence (excitation: 490 nm, emission: 515 nm). Therefore, Calcein-AM only stains live cells. On the other hand, 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 cell nucleus, and is embedded in the cell’s DNA double helix to generate red fluorescence (excitation: 535 nm, emission: 617 nm). 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. Due to the different optimal staining conditions for different cell lines, we recommend determining the appropriate concentration of Calcein-AM and PI individually.

I. Reagent   Calcein-AM 2mM 50uL in DMSO;  PI (1.5mM) 150uL in water

II. Observe cell morphology with a fluorescence microscope

Take HeLa cell staining as an example. Please note that different cell types and different concentrations have different observation conditions. According to the cell conditions, explore the best conditions for cell adhesion and reagent concentration under different conditions.

1. Preparation of dyeing solution

1) Dissolve 2 mg of Calcein-AM with 1 ml of anhydrous DMSO to prepare a 2 mmol/l Calcein-AM stock solution, and store it in a tightly sealed manner at -20°C.

2) Dissolve 2 mg of PI with 1 ml of ddH2O to prepare a 3 mmol/l PI stock solution. Store it in a tightly sealed manner at -20°C, which can be stored for one year.

3) Put the Calcein-AM stock solution and PI stock solution at room temperature.

4) Add 5 µl Calcein-AM stock solution and 15 µl PI stock solution to 5 ml PBS to prepare a staining solution. The final concentration of Calcein-AM is 2 µmol/l and the final concentration of PI is 4.5 µmol/l.

2. Cell staining

1) When staining adherent cells such as HeLa cells, first digest the cells with Trypsin-EDTA, etc. to prepare a cell suspension.

2) Centrifuge the cell suspension for 3 minutes (1,000 rpm).

3) Remove the supernatant, add PBS buffer, and adjust the number of cells to 10e5-10e6 cells/ml. Mix thoroughly with a pipette.

4) Since the serum in the culture medium contains esterase, Calcein-AM will decompose when exposed to water, which will cause the blank to rise, so it needs to be centrifuged several times and washed several times with PBS until it is completely washed.

5) Transfer 200 µl of cell suspension to a small test tube, add 100 µl of staining solution, and incubate at 37°C for 15 minutes.

6) Drop an appropriate amount of stained cell solution on the cover glass.

7) Under a fluorescence microscope, first excite with a wavelength of 490±10 nm to observe the yellow-green live cells, and also observe the red dead cells at the same time, and then excite with the 545 nm wavelength to see the red dead cells.

3. Optimal concentration of staining reagent

The optimal concentration of Calcein-AM and PI depends on different cell types. Through the following operations, we can find the optimal concentration of different cell staining reagents.

1) Prepare dead cells by incubating in 0.1% saponin or 0.1-0.5% digitonin for 10 minutes or by incubating in 70% ethanol for 30 minutes.

2) Stain dead cells with 0.1-10 µM PI solution to find the PI concentration that only stains the nucleus and not the cytoplasm.

3) Stain dead cells with 0.1-10 µM Calcein-AM solution to find the concentration of Calcein-AM that does not stain the cytoplasm. Then, live cells were stained with this concentration of Calcein-AM to check whether the live cells could be stained.

III. Matters needing attention

1) The ester part of Calcein-AM will decompose when exposed to moisture. After use, please keep it airtight and freeze at -20 degrees to prevent moisture from entering. When the Calcein-AM stock solution is diluted with buffer or culture medium, it should be prepared as soon as possible.

2) Always wear gloves, goggles, and masks when using. In case of contact with the skin, quickly wash with plenty of water.