DAPI solution DAPI染色液

生物活性

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。

本DAPI溶液用水配制，浓度为1mg/ml。

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.

注意事项：

1.DAPI对人体有一定刺激性，请注意适当防护。
2.荧光染料都存在淬灭的问题，建议染色后尽量当天完成检测。
3.为减缓荧光淬灭可以使用抗荧光淬灭封片液。
4.为了您的安全和健康，请穿实验服并戴一次性手套操作。

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化学性质

分子量	350.25
分子式	C16H15N5·2HCl
CAS号	28718-90-3
溶解性（25°C）	Water 1 mg/mL
储存条件	粉末型式       -20°C   3年；4°C   2年 溶于溶剂       -80°C   6个月；-20°C   1个月
运输方式	冰袋运输，根据产品的不同，可能会有相应调整。

储备液配制

*下述溶液配置方法仅为基于分子量计算出的理论值。不同产品在配置溶液前，需考虑其在不同溶剂中的溶解度限制。

Concentration / Solvent Volume / Mass	1 mg	5 mg	10 mg
1 mM	2.8551 mL	14.2755 mL	28.551 mL
5 mM	0.571 mL	2.8551 mL	5.7102 mL
10 mM	0.2855 mL	1.4276 mL	2.8551 mL

溶液配置摩尔浓度计算器

不同实验动物依据体表面积的等效剂量转换表（参考来源于公开文献）

	小鼠	大鼠	兔	豚鼠	仓鼠	狗
重量 (kg)	0.02	0.15	1.8	0.4	0.08	10
体表面积 (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km 系数	3	6	12	8	5	20

动物 A (mg/kg) = 动物 B (mg/kg) ×	动物 B的Km系数
	动物 A的Km系数

例如，依据体表面积折算法，将化合物用于小鼠的剂量20 mg/kg 换算成大鼠的剂量，需要将20 mg/kg 乘以小鼠的K_m系数（3），再除以大鼠的K_m系数（6），得到化合物用于大鼠的等效剂量为10 mg/kg。

参考文献

[1] Ruijun He, et al. HIF1A Alleviates compression-induced apoptosis of nucleus pulposus derived stem cells via upregulating autophagy

[2] Xia Zhang, et al. Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons

[3] Douglas S Grunwald, et al. GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction

[4] Paola Rusmini, et al. Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

[5] Israel C Nnah, et al. TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy