A Bulk Hemichannel Functional Level Detection Method Based on Fluorescent Dye Uptake

Abstract

A batch half-channel functional level detection method based on fluorescent dye uptake: (1) suspension culture, take THP-1 cells of a suspension culture cell line, use RPMI1640 culture medium supplemented with β-mercaptoethanol, and periodically add or replace the culture medium to protect cells The living environment is stable; monitor the concentration of the cell suspension does not exceed 1 × 10 6 cells / ml; the concentration of cell suspension was adjusted to 4×10 5 cells / ml, add tetradecanoyl phorbol acetate, inoculate the cell suspension in a 96-well plate, and change the medium after 24 hours to remove non-adherent cells; use blank RPMI1640 medium to maintain the culture to remove tetradecanoyl phorbol The effect of boitol acetate; (2) Fluorescent dye uptake to detect hemi-channel activity, ethidium bromide is a fluorescent dye, use standard RPMI1640 culture medium to prepare ethidium bromide fluorescent staining stock solution, and ethidium bromide staining stock solution The 96-well plate was added to the 96-well plate. After adding the fluorescent dye solution, the 96-well plate was placed on ice and incubated in the dark. The 96-well plate was placed in a fluorescence microplate reader to detect the fluorescence intensity.

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 of 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. Gap junction protein hexamers can form a channel-like structure on the cell membrane, and the gap junction protein hexamers of two adjacent cells are connected to form a gap junction intercellular communication (GJIC), and the selectivity allows less than Molecules or su...

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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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