अभिव्यक्ति और HEK-293T कक्ष में पुनः संयोजक वोल्टेज gated आयन चैनल के electrophysiological रिकॉर्डिंग के लिए अनुकूलित अभिकर्मक रणनीति

Summary

अत्यधिक कुशल के लिए विश्वसनीय विधि<em> इन विट्रो में</em> अभिव्यक्ति और पुनः संयोजक वोल्टेज gated सभ्य मानव भ्रूण गुर्दे की कोशिकाओं में आयन चैनल (HEK-293T) के बाद electrophysiological रिकॉर्डिंग.

Abstract

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28°C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28°C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37°C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28°C for further incubation. We find that the initial incubation at 28°C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Protocol

1. सेल संस्कृति है Dulbecco संशोधित ईगल्स मध्यम (DMEM, सिग्मा Aldrich) के 6mL युक्त मानव भ्रूण गुर्दे 293T कोशिकाओं (HEK-293T, Invitrogen) निकाल 25cm 2 (; Cellstar GREINER जैव – एक ऊतक इलाज संस्कृति) बोतल में पक्षपाती monolayers में बड़े हो रहे हैं 10 क…

Discussion

पूरे सेल में वर्णित प्रोटोकॉल और समाधान का उपयोग धाराओं रिकार्ड करने में सक्षम होने के नाते इंगित करता है कि अभिकर्मक सफल था और वांछित वोल्टेज gated आयन चैनलों या आयन चैनल परिसरों कोशिका झिल्ली में महत…

Materials

Material	Company	Catalogue Number	Comment
pIRES2-EGFP plasmid	Clontech	6029-1
Circle glass coverslips	Fisher Scientific	12-545-80	Circles No. 1 – 0.13 to 0.17mm thick, Size: 12mm
Amplifier	Axon Instruments	Axopatch 200B or Multiclamp 700B
Data Acquisition System	Axon Instruments	Digidata 1440A
Electrophysiology Software	Axon Instruments	pClamp10.1
Pipette manipulators	Sutter Instrument Co.	MPC-385-2
Epifluorescence inverted microscope	Zeiss Canada	Axiovert 40 CFL
Headstage	Axon Instruments	CV-7B
Headstage electrode holder	Axon Instruments	1-HL-U
Microelectrode holder for ground electrode	World Precision Instruments	MEH3RF 15
Electrophysiology capillary tubes	Sutter Instrument Co.	BF-150-86-15	Borosilicate glass with filament, O.D. 1.5mm, O.D. 0.86mm, 15cm long
Flaming/Brown Micropipette Puller	Sutter Instrument Co.	P-97
Micropipette fire polisher	Narishige	Micro Forge MF-830
Micro drug perfusion system	AutoMate Scientific	SmartSquirt8 Valvelink8.2
Drug perfusion system	AutoMate Scientific	Valvelink8.2 with Teflon valves