Batch half-channel function level detection method based on fluorescent dye uptake

Abstract

The invention relates to batch half-channel function level detection method based on fluorescent dye uptak. The method comprises the steps: (1) suspension culture: taking THP-1 cells of a suspension culture cell line, using an RPMI1640 culture solution added with beta-mercaptoethanol, and regularly adding or replacing the culture solution to ensure the steady state of the living environment of the cells; monitoring that the concentration of the cell suspension does not exceed 1 * 10 < 6 > / ml; adjusting the concentration of the cell suspension to be 4 * 10 < 5 > / ml, adding tetradecanoyl phorbol acetate, inoculating the cell suspension to a 96-well plate, and changing the solution 24 hours later to remove non-adherent cells; using a blank RPMI1640 culture solution for maintaining culture so as to remove the action effect of tetradecanoyl phorbol acetate; and (2) taking in a fluorescent dye to detect the activity of the semi-channel, taking ethidium bromide as the fluorescent dye, preparing an ethidium bromide fluorescent staining stock solution by using a standard RPMI1640 culture solution, adding the ethidium bromide fluorescent staining stock solution into a 96-well plate, adding the fluorescent staining solution, placing the 96-well plate on ice, incubating in a dark place, and placing the 96-well plate into a fluorescence microplate reader to detect the fluorescence intensity result.

Description

technical field [0001] The invention belongs to the field of protein channel structure and function research, in particular to a batch hemichannel function level detection method based on fluorescent dye uptake. Background technique [0002] Animal cells interact in a complex network formed by independent communication pathways, including direct (intercellular) cell-to-cell contacts and paracrine / autocrine (extracellular) signaling systems [EvansWH, E. DeVuyst, Leybaert L (2006). Thegap junction cellular internet: Connexin hemichannels enter the signaling limelight. Biochem J 397:1–14.]. This system is mainly realized by the specific functions of gap junction proteins widely expressed in various cells in the body. The gap junction protein hexamer can form a channel-like structure on the cell membrane, and the gap junction protein hexagrams of two adjacent cells are connected to form a gap junction intercellular communication (GJIC), and the selectivity is allowed to be less...

AI Technical Summary

Problems solved by technology

[0017] (1) It is difficult to reflect the real situation of the half-channel function level

[0018] Some key steps in traditional detection methods, such as washing cells with low calcium buffer before adding fluorescent dyes and adding dyes from a certain height through a pipette to apply mechanical stimulation, all open the hemichannels on the cell surface through external interference. Therefore, the results detected by the fluorescence intensity of the uptake dye reflect the relative number of hemichannels on the cell surface, but such detection ignores the key characteristic of hemichannels that are selectively open, and it is difficult to reflect the impact of the detected target stimulus on the cells. The true impact of hemichannel functional levels

[0019] (2) It is difficult to solve the confusion caused by intercellular communication junctions

However, there is a large detection bias in this scheme. First, the physiological state and function of cells grown alone are different from those grown in sheets, and may be more sensitive to stimulation. In addition, because they are not in contact with other cells, The channel protein originally used to form GJIC on the cell surface may also be detected as a hemichannel, resulting in inaccurate experimental results

[0021] (3) Complicated operation

[0022] Since the fluorescent dye uptake method has been around for a long time, the original intention of some operation steps is to target the characteristics of the fluorescent dyes used in the early days, but with the evolution of methods and dyes, it is no longer necessary, but it is still retained due to tradition, and detection requires the use of Fluorescence microscope takes pictures and uses image processing software to analyze, the overall operation process is very tedious and time-consuming

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