solution environments surrounding the patch-clamped cell. Precision switching ... Dynaflow platform improves the data qu
Dynaflow TM Application Note
Cell types & ion channels Typical experiment with Dynaflow Introduction In this note, a typcial dose response experiment is presented
Figure 1 Cell types
Ion channels
PC-12
Ligand gated
nAChr,α7
WSS-1
Ligand gated
nAChr,α2β3
RINm5F
Ligand gated
GABA A
L(-tk)
Ligand gated
ASIC
CHO
Ligand gated
hVR1
HEK
Ligand gated
Glycine
GH4C1
Ligand gated
5HT3
BC3H-1
Ligand gated
AMPA
N1E-115
Ligand gated
NMDA
COS 7
Ligand gated
P2X
neurons*
Ligand gated
Glutamate
The Dynaflow system has an extremely broad application
myocytes*
Voltage gated
KATP
base with regard to both cell types and ion channels [Figure 1].
*acutely isolated
Voltage gated
hERG
Cell types used with the system include typical mammalian
Voltage gated
hKv4.3
cell lines like CHO and HEK as well as larger cells (myocytes)
Voltage gated
hNav1.5
and cells with extensions (neurons). Ion channels studied
Voltage gated
hKv1.5
to illustrate the principles of running an experiment with Dynaflow. A brief introduction of utilized cell types and ion channels is followed by an explanation of the Dynaflow system background and the experimental set-up starting with the section GABA A expressed in HEK-agonist detection. A more detailed description of the filling of a Dynaflow mirofluidic chip and running a whole cell recording completes the experimental example.
Utilized cell types and ion channels
with the system range from very fast activating channels like
Figure 2
nACh and AMPA to ion channels requiring longer exposure times, like the hERG channel. The flexibility of the Dynaflow
control
system along with the stability of experimental parameters make it an ideal platform for conducting patch-clamp experiments.
Ligand gated ion channels The Dynaflow system is ideal when investigating ligand gated ion channels since it allows for rapid and precise switching of
500 pA
solution environments surrounding the patch-clamped cell. Precision switching enables investigations of receptor parameters such as rise and decay time, and receptor desensitization/resensitization kinetics. Recordings of entire
100 µM
100 ms
dose response curves from a single cell can easily be obtained with complete experimental control.
.c ell ectric on.c om Do wnl oad mor e applic ation not es: www Downl wnload more application notes: www.c .cell ellectric ectricon.c on.com GABAa high content recordings, Glutamate positive modulation, NMDA currents in acutely isolated neurons, hERG safety pharmacology screening and more.
The trace shows the recording of the current response during stimulation with a voltage pulse (+40mV for 1 second, 0.2Hz) and application of control (buffer flow) and 100 µM of antagonist. The antagonist exerts the major inhibitory effect by speeding up the inactivation kinetics and at high concentrations also by decreasing the peak current amplitude. More details on this experiment is given in the ”Inhibition of hKv4.3 in CHO-K1 cells” application.
Voltage gated ion channels
Figure 3
When studying voltage gated ion channels (Figure 3) 3), the Dynaflow platform improves the data quality and increases the number of data points that can be extracted from patchclamp recordings of a single cell. Forces acting on the cell due to the microfluidic flow have been shown to stabilize the seal and thereby prolong single cell recording times which is beneficial when long drug exposure times are required. The uniform flow from the micro channels facilitates an efficient wash out after drug application which can decrease total experimental time. Recordings with the Dynaflow system are highly reproducible, due
to the computer controlled
positioning of the cell in the laminar flow. For complicated mechanistic studies of ion channel function, Dynaflow has the advantage of synchronizing complex pulse protocols and sophisticated solution exchange events. Due to this experimental precision, data can be reliably obtained and analyzed with a minimized amount of variation.
Dynaflow system background The Dynaflow platform fully integrates with existing patchclamp equipment. The microfluidic system is automated and
The picture shows a recording of a GABAA recetor response to application of GABA at saturating concentration using the Dynaflow system. The vertical lines are the real time taggings indicating when the cell was passing the liquid boarder (eg the separting channel wall) between different solutions. The very precise solution exchange and ability to track these events in the recorded traces is one of the most important features of the Dynaflow system which makes it ideal for studies of ligand gated ion channels.
very easy to operate. Dynaflow drastically reduces experimental set-up and take-down time and allows for
Figure 4
reproducible execution of precise and robust patch-clamp experiments.
The system includes the microfluidic chip, a computer controlled scan stage used to translate chip movements, software to control and pre-program the movements of the scan stage, and a pump to drive the liquid flow. The Dynaflow Commander software synchronizes precision solution exchange with real time tagging in acquired data (Figure 2).
In Figure 4 a patch-clamped cell is shown positioned in the laminar flow zone in front of one of the microchannels. The liquid flow from the channel can be distinguished from the neighboring channels as being slightly darker. Colored solutions were used in this instance for the purpose of illustrating the distinct solution zones in the laminar flow.
GABAA expressed in HEK - agonist detection The dose response relationship of gamma-aminobutyric acid (GABA) to the GABAA receptor stably expressed in HEK cells was investigated using the Dynaflow platform and a DF-16 chip. The cell was patch-clamped in the whole cell configuration with a holding potential of -60 mV.
Figure 4 shows a patch-clamped cell is shown positioned in the laminar flow zone in front of one of the microchannels. The liquid flow from the channel can be distinguished from the neighboring channels as being slightly darker. Colored solutions were used in this instance for the purpose of illustrating the distinct solution zones in the laminar flow
DF-16 filling
Figure 5
Figure 5 is a cartoon of a Dynaflow 16 (DF-16) chip. The channel outlet area into the recording chamber is shown in a zoom in. Note the channel numbering and the connection to the sample wells. This notation will be used in most application data documents to visualize the solution content of each liquid
4
5
12
13
3
6
11
14
2
7
10
15
1
8
9
16
stream coupling to a specific ion channel response. In this picture the definition of positive (channel 1-16) and negative (channel 16-1) scanning direction is illustrated.
A DF-16 fill chart designed in Dynaflow Protocol Editor can be seen in Figure 6. The content of each well is included in the fill chart and automatically linked to an attached experimental protocol. The fill chart can be printed and saved for experimental documentation. On can also use the fill chart as a diagram to fill the chip properly.
In this experiment the even numbered channels of a DF-16 were filled with 80 µL of GABA at concentrations ranging from 1-100µM. Odd numbered channels were filled with extracellular buffer for washing the cell following GABA
Protocol Editor. A screen shot of this protocol and the Dynaflow Commander can be seen in Figure 7. Exposure time to GABA was pre-programmed to be 100 milliseconds followed by a 3 second buffer wash. Design and modification of protocols in the Protocol Editor is very fast and intuative. Protocols can be saved and printed allowing for efficient experimental logging.
Figure 6
Figure 7
CH 16
CH 13 CH 14 CH 15
CH 7 CH 8 CH 9 CH 10 CH 11 CH 12
Cell exposure times were pre-programmed in the Dynaflow
Positive scanning direction
CH 1 CH 2 CH 3 CH 4 CH 5 CH 6
DF-16 running
CH = Dynaflow chip channel
exposure.
Negative scanning direction
CH 13
CH 12
CH 11
CH 10
CH 9
CH 8
CH 7
CH 6
CH 5
CH 4
CH 3
Figure 9 CH 2
CH 1
Figure 8
CH 1
buffer
CH 2
1 µM
CH 3
buffer
CH 4
5 µM
CH 5
buffer
CH 6
10 µM
CH 7
buffer
CH 8
20 µM
CH 9
buffer
Figure 8 shows the peak current response of one cell scanned
CH 10
50 µM
over all concentrations of GABA in the positive scanning
CH 11
buffer
direction. Once the recording was initiated, obtaining the
CH 12
100 µM
Experimental results
complete dose response for this cell took only 30 seconds. The precision of solution exchange and exposure times with
buffer CH 14
CH 16
Dynaflow is clearly evident in the continuous trace shown in
buffer
Figure 8 8. Such experimental reproducibility allows for
buffer
obtaining recordings with very little variability.
buffer
Peak current responses from the mean of six cells were
CH = Dynaflow chip channel
plotted as a function of the logarithm of GABA concentration and fit to a Hill sigmoid function showing small standard
CH 16 - 1
CH 1 - 16
CH 16 - 1
CH 1 - 16
CH 16 - 1
CH 1 - 16
CH 16 - 1
CH 1 - 16
CH 16 - 1
deviation around the mean (Figure 9] 9]. CH 1 - 16
CH 16 - 1
CH 1 - 16
Figure 10
Figure 10 shows a set of 12 dose response curves obtained from one cell and illustrates how reproducible peak current response are with the Dynaflow platform. Using Dynaflow, it is easy to extract large amounts of relevant and high quality data from a patch-clamped cell.
References A Cell-Based Bar Code Reader for HighThroughput Screening of Ion Channel-Ligand Interactions J. Sinclair, J. Pihl, J. Olofsson, M. Karlsson, K. Jardemark, D.T. Chiu, and O. Orwar
Analytical Chemistry; Dec 15, 74 74(24), 61336138, 2002
Stabilization of High-Resistance Seals in Patch-Clamp Recordings by Laminar Flow
Analytical Chemistry; 75 75(23), 6718-6722, 2003
US:
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(Technical Note)
Rev. 1. 11/2006
J. Sinclair, J. Olofsson, J. Pihl, and O. Orwar
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