CN103571819B - Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability - Google Patents
Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability Download PDFInfo
- Publication number
- CN103571819B CN103571819B CN201310473138.9A CN201310473138A CN103571819B CN 103571819 B CN103571819 B CN 103571819B CN 201310473138 A CN201310473138 A CN 201310473138A CN 103571819 B CN103571819 B CN 103571819B
- Authority
- CN
- China
- Prior art keywords
- amphotericin
- fluid containing
- ivermectin
- quick
- intracellular fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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:
The invention belongs to biological field, be specifically related to a kind ofly to increase permeability of cell membrane fast thus fast rupture of membranes forms the method for high resistance seals.
Background technology:
The basic skills of patch clamp technique is attached to cell surface by glass microelectrode (tip diameter is at micron order), applies negative pressure and make to be formed between electrode and cell high resistance seals (namely 10
9the tight sealing-in of Europe rice magnitude).In an experiment, in glass electrode, specific antibiotic is added in liquid, as amphotericin B, membrane perforation can be made, intracellular fluid is communicated with liquid in electrode, and the electrical potential difference in the glass electrode be recorded to like this and between body lotion residing for cell is the film potential of cell, Here it is Perforated patch clamp.Perforation whole-cell patch-clamp effectively avoids soil washing, and kytoplasm seepage is very slow, can long-time stable record; There is the advantage little to cellular damage simultaneously.The duct that antibiotic perforation is formed, molecular diameter be greater than the ion of 0.8nm and molecule not by, and all ions all by, these ducts do not have voltage-dependent, make recording process by the impact of electrode command potential simultaneously.
Amphotericin B a kind ofly has antimycotic agent that is antibacterial or bactericidal action.Mainly change permeability of cell membrane by the ergosterol acted on cell membrane.Although Perforated patch clamp has so many advantages, the substantially water insoluble solution of amphotericin B, has therefore been with certain difficulty to Perforated patch clamp experiment.Also make Perforated patch clamp test seldom to use.
Ivermectin (IVERMECTIN) is the antiparasitic agent of a kind of New-type wide-spectrum, efficient, low toxicity, and this macrocyclic lipoid class medicine effectively can insert cell membrane increases membrane passage, but long duration of action can cause cytolysis.
Summary of the invention:
The object of this invention is to provide a kind of quick increase permeability of cell membrane thus the method for quick rupture of membranes formation high resistance seals.
Quick increase permeability of cell membrane of the present invention thus fast rupture of membranes form the method for high resistance seals, it is characterized in that, extracellular fluid containing ivermectin is poured in Quick-adding medicine pipe, again the intracellular fluid containing anphotericin is poured in microelectrode, then after utilizing the microelectrode clamp cells containing the intracellular fluid containing anphotericin to reach high resistance seals, mention attached cell to the Quick-adding medicine mouth of pipe, open Quick-adding medicine pipe, cell is fostered in containing the extracellular fluid of ivermectin, make cell Ra value under electro physiology test mode be down to 10, then stop fostering;
The described intracellular fluid containing anphotericin is prepared by the following method: be first dissolved in DMSO by amphotericin B, be mixed with the DMSO solution of 20mg/mL amphotericin B, then liquid in the DMSO solution of amphotericin B, the SDS solution of 100mmol/L and regular growth is mixed according to the ratio of volume ratio 50:15:1000, obtain the intracellular fluid containing 1mg/mL amphotericin B thus, be the intracellular fluid containing amphotericin B;
The described extracellular fluid containing ivermectin is dissolved in by ivermectin in the outer liquid of regular growth, makes ivermectin final concentration be 2mM/L, obtain the extracellular fluid containing ivermectin thus.
The cell that Using such method obtains can in 3min rapid solution cell membrane, simultaneously the amphotericin B of electrode interior continues to interact with the ergosterol in cell membrane and maintains cell membrane and be in stable permeability status for a long time, can be recorded to stable results of weak current in 1-2h.And record will be caused to interrupt because of cell membrane repeatedly closed in recording process.
In described regular growth, liquid is for containing 130mM CsMeth, 24mM CsCl, 10mM HEPES, 1mM CaCl
2, 1mM MgCl
2, solvent is water.
The outer liquid of described regular growth is for containing 154mM NaCl, 10mM D-glucose, 10mM HEPES, 1mMCaCl
2, 1mM MgCl
2, solvent is water.
The described intracellular fluid containing amphotericin B is prepared preferably by following methods:
The amphotericin B of every 0.01g is dissolved in 500mL DMSO, be placed on turbula shaker with 3200rpm rotating speed concussion 2min, be placed on after mixing in Ultrasound Instrument with the frequency ultrasound 5min of 40KHz, finally be mixed with the DMSO solution of 20mg/mL amphotericin B, again by the DMSO solution often drawing 50 μ L amphotericin Bs, in the regular growth that the SDS solution of 15 μ L100mmol/L is dissolved in 1mL in liquid, be placed on turbula shaker and shake 1min, then with the frequency ultrasound 10min of 40KHz, finally finding without precipitate suspended particles with the centrifugal 30s of the rotating speed of 500rpm, the existence of suspended particulate cause current loop not smooth because may block the micron-sized opening of glass electrode, record data are unstable, obtain the intracellular fluid containing amphotericin B thus.
The impact of amphotericin B cell membrane permeability has concentration dependent, increase membrane passage fast in conjunction with ivermectin while how allowing amphotericin B be dissolved in intracellular fluid to greatest extent, reach the shortest time and the best penetrate effect with this solve Perforated patch clamp application subject matter.The present invention solves the solubility of amphotericin B in intracellular fluid, it is made to be dissolved in preferably in intracellular fluid, in whole experimentation, amphotericin B is in water dissolved state, increase cell membrane penetration characteristic in conjunction with ivermectin, experiment starts preincubation cell and assists amphotericin B rupture of membranes at short notice together simultaneously.Stop after rupture of membranes fostering, under the help of amphotericin B, stabilizing cell membrane is in stable permeability status simultaneously, thus enables cell in 1-2h, be in stable electric current recording status.
Therefore the present invention can dissolving amphotericin B rapidly and efficiently in intracellular fluid, and increase permeability of cell membrane fast in conjunction with ivermectin thus quick rupture of membranes forms high resistance seals and tests in patch clamp experiments.
Detailed description of the invention:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
The amphotericin B of 0.01g is dissolved in 500mL DMSO, be placed on vortex-genie 2T turbula shaker with 3200rpm rotating speed concussion 2min, to be placed in Ultrasound Instrument with the frequency ultrasound 5min of 40KHz after mixing, to be finally mixed with the DMSO solution of 20mg/mL amphotericin B.Draw the DMSO solution of 50 μ L amphotericin Bs, the SDS solution of 15 μ L100mmol/L is dissolved in liquid in 1mL regular growth (containing 130mM CsMeth, 24mM CsCl, 10mM HEPES, 1mMCaCl
2, 1mM MgCl
2solvent is water) in, be placed on turbula shaker and shake 1min, then with the frequency ultrasound 10min of 40KHz, finally find without precipitate suspended particles with the centrifugal 30s of the rotating speed of 500rpm, cause current loop smooth because the existence of suspended particulate may block the micron-sized opening of glass electrode, record data are unstable again, obtain the intracellular fluid containing 1mg/mL amphotericin B thus, be the intracellular fluid containing amphotericin B.
Then ivermectin is dissolved in the outer liquid of regular growth (containing 154mM NaCl, 10mM D-glucose, 10mMHEPES, 1mM CaCl
2, 1mM MgCl
2, solvent is water) in, make the concentration of ivermectin be 2mM/L, obtain the extracellular fluid containing ivermectin thus.
The extracellular fluid containing ivermectin of 10ml the present embodiment is poured in Quick-adding medicine pipe, again the intracellular fluid containing amphotericin B is poured in microelectrode, then after utilizing the microelectrode clamp cells containing the intracellular fluid containing amphotericin B to reach high resistance seals, mention attached cell to the Quick-adding medicine mouth of pipe (the dosing mouth of pipe containing the extracellular fluid of ivermectin), open Quick-adding medicine pipe.After cell fosters 2min in the extracellular fluid containing ivermectin, cell Ra value under electro physiology test mode is down to 10, stops fostering.The cell that Using such method obtains can in 3min rapid solution cell membrane, simultaneously the amphotericin B of electrode interior continues to interact with the ergosterol in cell membrane and maintains cell membrane and be in stable permeability status for a long time, stable results of weak current can be recorded in 1-2h, record will be caused to interrupt because of cell membrane repeatedly closed in recording process.
Claims (4)
1. one kind increases permeability of cell membrane thus the method for quick rupture of membranes formation high resistance seals fast, it is characterized in that, extracellular fluid containing ivermectin is poured in Quick-adding medicine pipe, again the intracellular fluid containing amphotericin B is poured in microelectrode, then after utilizing the microelectrode clamp cells containing the intracellular fluid containing amphotericin B to reach high resistance seals, mention attached cell to the Quick-adding medicine mouth of pipe, open Quick-adding medicine pipe, cell is fostered in containing the extracellular fluid of ivermectin, make cell Ra value under electro physiology test mode be down to 10, then stop fostering;
The described intracellular fluid containing amphotericin B is prepared by the following method: be first dissolved in DMSO by amphotericin B, be mixed with the DMSO solution of 20mg/mL amphotericin B, then liquid in the DMSO solution of amphotericin B, the SDS solution of 100mmol/L and regular growth is mixed according to the ratio of volume ratio 50:15:1000, obtain the intracellular fluid containing 1mg/mL amphotericin B thus, be the intracellular fluid containing amphotericin B;
The described extracellular fluid containing ivermectin is dissolved in by ivermectin in the outer liquid of regular growth, makes ivermectin final concentration be 2mmol/L, obtain the extracellular fluid containing ivermectin thus.
2. method according to claim 1, it is characterized in that, the described intracellular fluid containing amphotericin B is prepared by the following method: the amphotericin B of every 0.01g is dissolved in 500mL DMSO, be placed on turbula shaker with 3200rpm rotating speed concussion 2min, be placed on after mixing in Ultrasound Instrument with the frequency ultrasound 5min of 40KHz, finally be mixed with the DMSO solution of 20mg/mL amphotericin B, again by the DMSO solution often drawing 50 μ L amphotericin Bs, in the regular growth that the SDS solution of 15 μ L100mmol/L is dissolved in 1mL in liquid, be placed on turbula shaker and shake 1min, then with the frequency ultrasound 10min of 40KHz, finally find without precipitate suspended particles with the centrifugal 30s of the rotating speed of 500rpm again, obtain the intracellular fluid containing amphotericin B thus.
3. method according to claim 1 and 2, is characterized in that, in described regular growth, liquid contains 130mM CsMeth, 24mM CsCl, 10mM HEPES, 1mM CaCl
2, 1mM MgCl
2, solvent is water.
4. method according to claim 1 and 2, is characterized in that, the outer liquid of described regular growth contains 154mM NaCl, 10mM D-glucose, 10mM HEPES, 1mM CaCl
2, 1mM MgCl
2, solvent is water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310473138.9A CN103571819B (en) | 2013-10-11 | 2013-10-11 | Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310473138.9A CN103571819B (en) | 2013-10-11 | 2013-10-11 | Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103571819A CN103571819A (en) | 2014-02-12 |
| CN103571819B true CN103571819B (en) | 2015-03-25 |
Family
ID=50044533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310473138.9A Expired - Fee Related CN103571819B (en) | 2013-10-11 | 2013-10-11 | Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103571819B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911241B (en) * | 2015-05-27 | 2018-06-19 | 中国科学院广州生物医药与健康研究院 | A kind of intracellular fluid and its method of quick increase permeability of cell membrane |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059447A1 (en) * | 2000-02-11 | 2001-08-16 | Yale University | Planar patch clamp electrodes |
| WO2004018690A1 (en) * | 2002-08-21 | 2004-03-04 | Cellectricon Ab | System and method for obtaining and maintaining high resistance seals in patch clamp recordings |
| US6776896B1 (en) * | 2000-10-11 | 2004-08-17 | Axon Instruments, Inc. | Method of positioning cells for electrophysiological testing |
| US7521255B2 (en) * | 2004-02-26 | 2009-04-21 | Commissariat A L'energie Atomique | Method and device for controlling the positioning of a biological element on a support |
| CN102071135A (en) * | 2009-11-20 | 2011-05-25 | 国家纳米技术与工程研究院 | High resolution patch clamp based on scanning probe microscopy technology and operating method thereof |
-
2013
- 2013-10-11 CN CN201310473138.9A patent/CN103571819B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059447A1 (en) * | 2000-02-11 | 2001-08-16 | Yale University | Planar patch clamp electrodes |
| US6776896B1 (en) * | 2000-10-11 | 2004-08-17 | Axon Instruments, Inc. | Method of positioning cells for electrophysiological testing |
| WO2004018690A1 (en) * | 2002-08-21 | 2004-03-04 | Cellectricon Ab | System and method for obtaining and maintaining high resistance seals in patch clamp recordings |
| US7521255B2 (en) * | 2004-02-26 | 2009-04-21 | Commissariat A L'energie Atomique | Method and device for controlling the positioning of a biological element on a support |
| CN102071135A (en) * | 2009-11-20 | 2011-05-25 | 国家纳米技术与工程研究院 | High resolution patch clamp based on scanning probe microscopy technology and operating method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 穿孔膜片钳技术在离子通道电流研究中的应用;李鹏云等;《四川生理科学杂志》;20060630;第28卷(第02期);第62-65页 * |
| 膜片钳技术及其应用;杜育哲等;《天津农学院学报》;20010630;第8卷(第02期);第24-26页 * |
| 酚噻嗪对豚鼠心室肌细胞动作电位时程和跨膜离子电流作用的研究;王海昌等;《中国心脏起搏与心电生理杂志》;20030625;第17卷(第03期);第211-216页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103571819A (en) | 2014-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fontana et al. | Three-dimensional structure of Rubella virus factories | |
| Gao et al. | Tension-induced vesicle fusion: pathways and pore dynamics | |
| CN103567431B (en) | A kind of nano-class silver colloidal solution and preparation method thereof | |
| WO2014172045A8 (en) | Lipid nanoparticles for transfection and related methods | |
| CN102899272A (en) | Method for inducing bacteria to enter viable but nonculturable state | |
| CN103571819B (en) | Method for forming high-resistance seal with quick rupture of membrane by quickly improving membrane permeability | |
| JP2013531975A (en) | Dynamic mixing, electroporation chamber and system | |
| JP2016503315A5 (en) | ||
| Vancini et al. | Alphavirus entry into host cells | |
| Nablo et al. | Anthrax toxin-induced rupture of artificial lipid bilayer membranes | |
| WO2008100749A3 (en) | Transfection in magnetically driven continuous flow | |
| CN103372382B (en) | A kind of preparation facilities of therapeutic type micro air bubble ultrasonic contrast medium and method | |
| CN105168249B (en) | A kind of antimicrobial composite material graphene oxide-triclosan and preparation method thereof | |
| Dryzer et al. | Electropermeabilization of nematode eggs for parasite deactivation | |
| CN104911241A (en) | Intracellular fluid and method for rapidly increasing cell membrane permeability | |
| CN103397017B (en) | Formation method of inside-out patch in patch clamp experiment | |
| Kim et al. | Electro-responsive transdermal drug release of MWCNT/PVA nanocomposite hydrogels | |
| Abd Rahman et al. | Pulse Electric Field Effect on Breast Cancer Cell Properties | |
| Demming | Nanotechnological selection | |
| CN103893830B (en) | Nanometer silver infection hernia patch and preparation method thereof | |
| Tang et al. | Temperature-/CO 2-dual-responsiveness of a zwitterionic “schizophrenic” copolymer | |
| JP2011255319A (en) | Method for producing vesicles | |
| JP5585981B2 (en) | Photoresponsive liposome and method for transporting substance using photoresponsive liposome | |
| CN202305471U (en) | High-impedance sealing device for simple and convenient patch clamp | |
| Jiang et al. | Self-supporting pH-responsive Micro porous Membrane from Copolymer PVA-co-PDEAEMA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150325 Termination date: 20161011 |