DAPI Reagent (25μg/mL)
中文名称:4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色液

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检测原理 | DAPI(4',6-二脒基-2苯基吲哚)是一种发蓝色荧光的 DNA 染料,在与 dsDNA 的 AT 区结合时,荧光增强约20倍。DAPI 由紫色(405 nm)激光激发,通常在荧光显微术、流式细胞术和染色体染色中作为核复染剂。由于其光谱特性,DAPI 非常适合与绿色(Invitrogen Alexa Fluor 488, FITC, GFP)和红色(Invitrogen Alexa Fluor 594,rhodamine,Invitrogen Texas red, mCherry, mKate-2)荧光基团在多色实验中共用。 |
Ex/Em | 410/450 |
Channel set | Pacific Blue |
保存温度 | 该产品可在 2~8°C避光条件下保存12个月。 |
有效期 | 12个月 |
运输条件 | 冰袋 |
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Q1:我的样本需要做活死染料来排除死细胞嘛?不用会有什么影响?
死细胞的自发荧光会很强烈,如果不用死活染料排除死细胞,可能会在分析结果的散点图中出现沿着对角线分布的细胞群,影响实验结果。所以为了确保实验结果的准确性,对于除脾脏外的组织样本,一般建议加死活染料来排除死细胞。如果是血液样本,如果样本新鲜,并且只做少数几个细胞表面染色的多染,也可以不排除死细胞,但这需要预实验确定样品中死细胞不多或者不会对结果造成干扰。
可通过对现有数据的死细胞群体进行分析,看看这些细胞在荧光通道的分布以及对应的比例是不是和活细胞群体基本一致,如果一致,可以不做死活细胞染色,不一致的话,建议加上活死染料。
Q2:我想检测肿瘤相关巨噬细胞,只用F4/80检测可以吗?
建议老师加上CD45、CD11b和死活染料。一方面,肿瘤样本中细胞种类多且巨噬细胞比例较低,很难找到目标群。另一方面,死细胞容易摄取抗体和探针,导致明显的非特异性染色,而且死细胞自发荧光特别强,会导致背景荧光增强,使得我们无法观察到一些标记的弱阳性表达。所以建议老师通过死活染料排除死细胞干扰,然后圈CD45阳性找到目的细胞群,最后通过CD11b和F4/80确定巨噬细胞比例。
