Intracellular pH value detection method

Abstract

The invention relates to the field of single cell detection, in particular to an intracellular pH value (pHi) detection method. The method comprises the following steps: (1) co-incubating to-be-detected cells with a cell membrane-permeable and pH-sensitive fluorescent dye, and putting the to-be-detected cells loaded with the fluorescent dye into a buffer solution in an air and temperature controldevice; with the assistance of a perfusion / drug delivery system, using a device capable of monitoring the fluorescence change of cells in real time to record the cell fluorescence intensity data corresponding to different treatments; (2) treating the cells with calibration solutions of different known pH values, establishing a standard curve of cell fluorescence intensity-cell pHi related data, and substituting cell fluorescence intensity data into the standard curve to obtain a single cell pHi. The method can monitor the change of pHi of single or multiple cells for a long time and in real time without damaging the cells, has good reproducibility, and is suitable for detecting the pH of cells of various sizes.

Description

technical field [0001] The invention relates to the field of single cell detection, in particular to a method for detecting intracellular pH. Background technique [0002] pH is a measure of the activity of protons (tiny charged particles that attach to other molecules). Real-time detection of pH in living biological systems is crucial for detecting and understanding the impact of pH on relevant physiological conditions (such as the study of ion channels on the cell surface, the study of certain receptors, or the study of cell development, etc.). important. There are few reports in China about the real-time monitoring technology of pH value (pHi) in a single cell. At present, there are roughly three methods for detecting pHi in other countries in the world. [0003] 1. Weak acid and weak base distribution method: firstly select a suitable radioactive weak acid (or weak base) to treat the cells according to the needs. Because weak acids (or weak bases) have membrane perme...

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Problems solved by technology

[0007] 1. Weak acid and weak base distribution method: It takes a long time to equilibrate, and the real-time change of pHi value cannot be measured; in addition, some weak acids and weak bases cause great damage to cells, which makes the measurement results biased

[0008] 2. NMR method: It needs to maintain the stable physiological state of cells for a long time, and the time resolution is low; it needs a large number of cells to measure, and it is not suitable for the determination of the pHi value of a single cell; it is not suitable for the determination of acidic (pH7.5) cell pHi value

[0009] 3. Microelectrode method: The technical operation is difficult, and it is necessary to accurately grasp the position of the electrode insertion; and it is easy to cause loss to the inserted part of the cell and cause ion leakage; it is not suitable for the determination of the pHi value of small-diameter cells

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