Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Samenvatting

Source: Grimm, C., et al. Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins. J. Vis. Exp. (2017)

In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

Protocol

1. Setup Prior to Recordings

1. Prepare the recording solutions with ion concentrations as indicated in Table 1.​
   1. Prepare 15 mL of 2 M stock solutions in ultrapure water for MgCl₂, CaCl₂, KCl and CsCl.
   2. Weigh the solid components for 100 mL of the intracellular and 500 mL of the extracellular buffered solutions according to Table 1 into separate beakers and add the respective amount of the prepared stock solutions afterward. For example, for the intracellular buffer, use 0.3803 g (10 mM) EGTA(ethylene glycol tetraacetic acid), 0.2383 g (10 mM) HEPES(4-(2-hydroxyethyl)-1-pipera....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
HEK293 cells	Sigma Aldrich	85120602	Human embryonic kidney cells
Retinal	Sigma Aldrich	R2500	all-trans retinal
FuGENE HD	Promega	E2312	Transfection reagent
DMEM	Biochrome	FG 0445	Dulbecco's Modified Eagle Medium
Agarose	Roth	3810	Agar bridges
CaCl2	Roth	5239	CaCl2 2H2O
CsCl	Biomol	2452
EGTA	Roth	3054
FBS	Biochrome	S0615	Cell culture
Glucose	Roth	HN06	D(+)-Glucose
KCl	Roth	6781
MgCl2	Roth	2189	MgCl2 6H2O
NaCl	Roth	3957
NMG	Sigma Aldrich	M2004	N-Methyl-D-glucamine
Na-Aspartate	Sigma Aldrich	A6683	L-Aspartic acid sodium salt monohydrate
Citric acid	Roth	6490
AgeI	ThermoFischerScientific	ER1462	Restriction enzyme
XhoI	ThermoFischerScientific	ER0695	Restriction enzyme
NheI	ThermoFischerScientific	ER0975	Restriction enzyme
XL1Blue E.coli/	Agilent Technologies	200249	Chemocompetent E.coli
Kanamycin	Roth	T832
Lysogeny broth medium	Roth	X964
Agar-Agar	Roth	6494	Agar plates
Plasmid purification kit	Marchery-Nagel	740727.25
Penicilin/Streptomycin	Biochrome	A 2213	Cell culture
Poly-D-lysine hydrobromide	Sigma Aldrich	P6407-5MG	Cover slip coating
Microforge	Custom made		Fire polishing
Serological pipettes	TPP		Different sizes
Clean bench	Kojair	Biowizard SL130
Stirrer	IKA	RCT classic
Silver wire	Science Products	AG-T25; AG-T10	Electrodes, 0.64 mm (bath); 0.25 mm (electrode)
pH-meter	Knick	765 Calimetric
Osmometer	Vogel	OM 815
Microscope	Carl Zeiss	ID03	Fire polishing
CO2 incubator	Binder	CB150
Cell culture dishes	TPP	93040	34 mm internal diameter
Cover slips	Roth	P232	15 mm diameter
Thermometer	Rössel Messtechnik	MTM12
Beamsplitter	Chroma	21011	90/10 transmission
Pipette holder	ALA Scientific Instruments	PPH-1P-AXU-0-1.5
Headstage	Molecular Devices	CV203BU
Amplifier	Molecular Devices	AxoPatch200B
Digitizer	Molecular Devices	DigiData1400	Digital analog converter
Lightsource	TILL Photonics	Polychrome V	Set to 540 nm full intensity
Microscope	Carl Zeiss	Axiovert 100
Shutter	Vincent Associates	VS25
Shutter driver	Vincent Associates	VCM-D1
Glass capilarries	Warner Instruments	G150F-3	Boresilicate capillaries with fire polished ends OD 1.5 mm ID  0.86 mm
Micropipette puller	Sutter Instruments	P1000
Bath handler	Lorenz Messgerätebau	MPCU
Tripleband filterset	Chroma	69008	Fluorescence filter  ECFP/EYFP/mCherry
CCD camera	Watec	Wat-221SCCD
Optometer	Gigahertz Optik	P9710	Measure light intensities
Objective	Carl Zeiss	421462-9900-000	W Plan-Apochromat 40X/1.0 DIC
Micromanipulator	Scientifica	PatchStar
Recording chamber	Custom made
Power supply	Manson	HCS-3202	Avoids electrical noise from microscope built-in power supply
Vibration isolated table	Newport	M-VW-3636-OPT-01
Faraday cage	Custom made or any commercial matching table
Hoses	Any comercial; e.g. Roth		Different sizes and materials for bath handling and application of pipette pressure; agar bridges
Linear shaker	Sunlab Instruments	SU 1000
Liquid junction potential calculator	Molecular Devices or directly from Peter H. Barry		Program is included in the Clampex aquisition software or can be obtained from  p.barry@unsw.edu.au
Data acquisition software	Molecular Devices	Clampex 10.X
Data evaluation software	Molecular Devices	Clampfit 10.X
PsACR1	GenBank or Addgene	KF992074.1 or Addgene plasmid #85465	Gene encoding for PsACR1
Amplifier guide	Molecular Devices	The Axon Guide