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Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability

A high-resistance sealing and permeability technology, used in biochemical equipment and methods, electric/wave energy treatment of enzymes, and electric/wave energy treatment of microorganisms, etc., can solve problems such as cell lysis and increase cell membrane permeability. sexual effect

Inactive Publication Date: 2014-02-12
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0004] Ivermectin (IVERMECTIN) is a new type of broad-spectrum, high-efficiency, and low-toxic antiparasitic drug. This macrocyclic lipid drug can effectively insert into the cell membrane to increase the permeability of the cell membrane, but long-term action will cause cell to dissolve

Method used

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Examples

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Embodiment 1

[0018] Dissolve 0.01g of amphotericin B in 500mL DMSO, place it on a vortex-genie 2T vortex shaker at 3200rpm for 2min, mix it and put it in an ultrasonic instrument at a frequency of 40KHz for 5min, and finally prepare 20mg / mL amphotericin B in DMSO. Pipette 50 μL amphotericin B in DMSO solution, 15 μL 100 mmol / L SDS solution and dissolve in 1 mL conventional intracellular solution (containing 130 mM CsMeth, 24 mM CsCl, 10 mM HEPES, 1 mM CaCl 2 ,1mM MgCl 2 , the solvent is water), placed on a vortex shaker for 1min, then ultrasonicated at a frequency of 40KHz for 10min, and finally centrifuged at a speed of 500rpm for 30s, no suspended particles were found, because the existence of suspended particles may block the glass electrode micron The opening of the opening causes the current circuit to be unsmooth, and the recorded data is unstable, thus obtaining the intracellular fluid containing 1 mg / mL amphotericin B, that is, the intracellular fluid containing amphotericin B. ...

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Abstract

The invention discloses a method for forming high-resistance seal with quick rupture of a membrane by quickly improving membrane permeability. The method comprises the following steps: infusing an extracellular fluid containing ivermectin into a quick chemical feed pipe, then infusing an intracellular fluid containing amphotericin into a microelectrode, thereafter clamping cells by utilizing the microelectrode of the intracellular fluid containing amphotericin to realize high-resistance seal, lifting the anchorage-dependent cells to the opening of the quick chemical feed pipe, opening the quick chemical feed pipe to culture the cells in the extracellular fluid containing ivermectin and reduce the Ra value of the cells to 10 at an electrophysiological test state, and then ending culturing. The method disclosed by the invention can be used for quickly and efficiently dissolving amphotericin B in the intracellular fluid and combining the amphotericin B with the ivermectin to quickly improve the membrane permeability, so that quick rupture of the membrane is realized in the membrane clamp test to form high-resistance seal for experiments.

Description

Technical field: [0001] The invention belongs to the field of biology, and in particular relates to a method capable of rapidly increasing cell membrane permeability so as to quickly rupture the membrane and form a high-resistance seal. Background technique: [0002] The basic method of the patch clamp technique is to attach a glass microelectrode (tip diameter in the micron order) to the surface of the cell, and apply negative pressure to form a high-resistance seal between the electrode and the cell (ie, 10 9 ohm-level tight seal). In the experiment, adding specific antibiotics, such as amphotericin B, to the inner liquid of the glass electrode can perforate the cell membrane, and the inner liquid of the cell and the inner liquid of the electrode are connected, so that the recorded glass electrode and the bath liquid where the cell is located The potential difference is the membrane potential of the cell, which is the perforated patch clamp. The perforated whole-cell pat...

Claims

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Application Information

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IPC IPC(8): C12N13/00
Inventor 李志远梁欣尹世奎
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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