DAPI solution  别名：4',6-Diamidino-2-phenylindole dihydrochloride; DAPI染色液

DAPI是一种可对DNA 染色的细胞核染色试剂，它在嵌入双链DNA后释放蓝色荧光。DAPI常用于细胞凋亡检测，染色后用荧光显微镜观察或流式细胞仪检测。尽管DAPI不能通过活细胞膜，但却能穿透扰乱的细胞膜而对核染色。DAPI具有很高的光漂白承受水平，能用来检测酵母线粒体DNA，叶绿体DNA，病毒DNA，microplasm DNA以及染色体DNA。DAPI-DNA复合物的激发和发射波长分别为360nm和460nm。

用于细胞核染色时，推荐的DAPI工作浓度为0.5-10μg/ml。

Protocols for Counterstaining with DAPI:

Immunofluorescence Staining
Note: Cells may be fixed by method of choice, such as with 4% paraformaldehyde. DAPI staining is done after staining for other markers. Fixation and permeabilization of cells is not necessary to counterstain with DAPI.

1. Fix cells using method of choice.
2. Incubate the cells with phosphate buffered saline (PBS) for 15 minutes.
3. Dilute the DAPI solution (we recommend to 300 nM) with PBS. Remove excess PBS from the slide and cover with DAPI solution, making sure the cells are completely covered.
4. Incubate for up to 5 minutes. Rinse the slide several times to remove all free DAPI. It is recommended to use a mounting medium with antifade reagent to reduce fluorescence quenching. The slide is ready to view under the microscope with appropriate filters.


Nuclear Staining for Flow Cytometry
Note: Cells may be fixed with 4% paraformadehyde or absolute ethanol.

1. Fix cells with absolute ethanol or 4% paraformaldehyde.
2. Centrifuge, discard the supernatant, and add 5 ml of PBS, allowing cells to rehydrate for 15 minutes.
3. Dilute the DAPI to 3 µM (recommended) in staining buffer.
4. Centrifuge cell suspension from step 3 above, discard supernatant, and add 1 ml of DAPI solution, then incubate at room temperature for 15 minutes.
5. Analyze by flow cytometry using an instrument with appropriate lasers.

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