Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed
Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed
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DOI：
10.1007/978-1-4939-6937-1_16
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发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Schrader, Michael
中科院分区：
文献类型：
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作者：
Godinho, Luis F.;Schrader, Michael
细胞器pH稳态对于维持适当的细胞功能至关重要。过氧化物酶体pH值的性质仍然有些争议，有几项研究报告了相互矛盾的结果。在这里，我们详细描述了一种快速，准确的方法用于测量过氧化物酶体pH值，使用pHRed传感器蛋白和共聚焦显微镜的活哺乳动物细胞。pHRed是一种pH的比率传感器，通过C-末端靶向序列靶向过氧化物酶体。该探针在610 nm处具有最大荧光发射，同时在440和585 nm处显示出双激发峰，允许在活细胞显微镜中进行比率成像和测定细胞内pH。
Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.