Cell potassium electrode property detection method based on Nernst electric potential fluorochrome

A technology of fluorescent dyes and electrode characteristics, applied in the biological field, can solve the problems of inapplicability, inconvenient promotion by ordinary research and development institutions, expensive instruments, etc.

Inactive Publication Date: 2012-03-28
CHONGQING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The limitations of applying the patch clamp technique to evaluate the characteristics of the cell potassium electrode are: ① the patch clamp technique is delicate in operation, expensive instruments, high technical requirements, and it is not easy to popularize in general research and development institutions; a cell
The above-mentio

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  • Cell potassium electrode property detection method based on Nernst electric potential fluorochrome
  • Cell potassium electrode property detection method based on Nernst electric potential fluorochrome
  • Cell potassium electrode property detection method based on Nernst electric potential fluorochrome

Examples

Experimental program
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Embodiment 1

[0040] Example 1 Establishment of a theoretical induction method for evaluating the characteristics of a cell potassium electrode

[0041] A theoretical analysis

[0042] For cells with the properties of a Nernst potassium electrode, the cell resting membrane potential V m The relationship with the concentration of potassium ions is described by the Nernst equation, namely:

[0043] V m = - 2.303 RT zF log [ K + ] i [ K + ] o - - - ( 1 )

[0044] Here R is the ideal gas constant; T is the absolute temperature; Z...

Embodiment 2

[0071] Example 2 Measurement and Evaluation of Cellular Potassium Electrode Characteristics

[0072] 1. Determination of intracellular sodium ion concentration in neural stem cells

[0073] Na in intracellular fluid + Relative content and concentration are low, available Na + Fluorescent dyes are used to measure. Commonly used Na + Fluorescent dyes include SFBI, Sodium Green, etc., and the sodium ion dye used in this example is CoroNa TM Green (Invitrogen, USA. Cat No.: C36676).

[0074] Experimental principle: In this example, after the cells are permeabilized with 18 μM monensin, the sodium ions inside and outside the cells will be balanced. In setting external Na + Under the condition of concentration, the cells are loaded with CoroNa Green, which can measure different Na + The fluorescence intensity corresponding to the concentration was used to make a standard curve. Under the same conditions, use the same concentration of CoroNa Green on Na + staining, the intr...

Embodiment 3

[0142] Example 3 Evaluation of Cellular Potassium Electrode Characteristics by Patch Clamp Technique and Verification of Potential Fluorescent Dye Method

[0143] 1. Materials and Reagents

[0144] Amphotericin B (domestic), trypsin-EDTA (Hyclone, Cat.No.: SH70042.01), the inner and outer solution of the electrode contains K + 135mM HBS (without Na + );

[0145] Instrument: EPC-10 single-channel patch clamp (HEKA Electronic, German) and related accessories

[0146] 2. Test steps

[0147] 1 The cell culture and inoculation methods were the same as in Example 2, digested with 0.125% trypsin-EDTA, observed under an inverted phase-contrast microscope, the cells were raised but not completely rounded, and the trypsin was discarded. with K + Rinse the dish with 5mM HBS and soak the cells. Divide into two groups and carry out test, valinomycin group and normal group (processing method is the same as embodiment 2).

[0148] 2. Pre-absorb 30 μg / ml amphotericin B at the tip of th...

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Abstract

The invention especially relates to a cell potassium electrode property detection method based on a Nernst electric potential fluorochrome. The method comprises specific steps that: cells with Nernst potassium electrode properties are established, [K+]i/f is obtained with a electric potential fluorochrome technology; intracellular sodium ion concentration is evaluated by using sodium ion fluorochrome; inside-to-outside fluorescence intensity ratio of the cells stained by using the electric potential fluorochrome is measured in a gradient potassium solution; f, [K+]i and a cell apparent sodium/potassium osmosis ratio PNa/K are obtained through non-linear iteration fitting according to a Goldman equation; and PNa/K serves as a quantitative index in the evaluation of cell potassium electrodeproperties. According to the invention, the cell potassium electrode property evaluation method provides a novel index and a novel method for researching cell-electricity biophysical properties and ionic channel function expression by using an electric potential fluorochrome technology, and provides possibilities of non-intrusion in-situ detections of cell-electricity biophysical properties and nerve cell functional differentiation under a material three-dimensional structure.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a method for detecting the characteristic evaluation value of a cell potassium electrode based on a Nernst potential fluorescent dye. Background technique [0002] The electrobiophysical or electrophysiological properties of cells are the direct manifestations of cell ion channel expression and functional differentiation or functional maturation, and are also important indicators for evaluating the function of some cells, especially excitable cells. Although the most commonly used and classical method for evaluating cell electrobiophysical or electrophysiological properties is still the glass microelectrode technology represented by patch clamp, the invasiveness of this method and the fact that under certain conditions (such as living tissue and three-dimensional structure of materials) The limitations of internal) applications have led to the gradual adoption of more practical...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N21/64
Inventor 吴泽志张利光宋兆全林雨罗美蓉黄岂平廖彦剑金良李晨钟威廉.S.克萨阿利达陈景龙
Owner CHONGQING UNIV
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