Intracellular pH value detection method

A detection method and cell technology, applied in the field of single-cell detection, can solve the problems of inappropriate single-cell pHi measurement, large cell damage, long equilibration time, etc.

Active Publication Date: 2018-09-14
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Abstract
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  • Claims
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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 n...

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Embodiment

[0048] 1. Collection of cumulus-oocyte complexes (COCs): After collecting animal ovaries, wash the ovaries with PBS solution, and quickly put them into a petri dish filled with ovum collection liquid to ensure that the ovum collection liquid submerges the ovaries. For small animal ovaries, the operator needs to hold a 1ml syringe with a 26G needle in both hands, one of which uses the syringe and needle to hold the ovary still, and the other hand uses the syringe and needle to puncture the protruding follicles on the surface of the ovary , thereby releasing COCs, and collecting COCs under a stereoscope. For the ovaries of large animals, you can hold the ovary with one hand, and with the other hand use a 10ml syringe connected with an 18G needle (containing a small amount of egg collection fluid to prevent COCs from sticking to the wall of the syringe cavity) to suck 3-8mm follicles, and place The collected liquid was placed under a stereoscope to collect COCs.

[0049] 2. Coll...

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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...

Claims

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Application Information

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IPC IPC(8): G01N15/10
CPCG01N15/10G01N15/1463G01N2015/1006
Inventor 许保增张宇飞杨镒峰魏海军李晓霞赵伟刚陈秀敏刁云飞王丽英常彤薛海龙王士勇曹俊国
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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