Nanion Technologies
...

Data and Applications


Acetylcolin Receptor

a3b4

Shown is a raw current response of a HEK293 cell expressing AChR (α3β4) to exposure to 300 μM nicotine. Solutions were  stacked (layered) in the pipette to achieve brief exposure times.

ACh Dose Response Curve (nAChR a7)

AChRCRC

Increasing acetylcholine concentrations (30 µM, 100 µM and 300 µM) were added to the same cell using a stacked applications protocol.
Cells were kindly provided by Galantos Pharma GmbH.

Activation of TRPV1 by Internal Application of Capsaicine

0TRPV1_Internal_Act

The SyncroPatch 96 allows continues recording during the application of compounds from the intracellular side. Here, TRPV1 channels were activated by the internal application of capsaicine.

Automated Analysis

SyncroPatch Analysis Tool

With the SyncroPatch Analysis Tool - DataControl96, IC50 plots are easily generated, displayed, averaged, evaluated, and modified.

Here, Lidocaine concentration response curves (CRC) of rNav1.8 expressing ND7-23 cells are shown.

Cells were kindly provided by EMD Millipore.

Blind Study on hNav1.5 Antagonists Using an Inactivation Protocol

NavInact

22 selected compounds were tested in a blind study on the SyncroPatch 96 using HEK 293 cells expressing hNav1.5 ion channels. The IC50-values were compared to manual patch clamp measurements, performed at the customer site.

Cav1.2 Current Voltage Relationship

Cav12_IV

Shown are raw current traces (top) and the constructed peak current-voltage relationship (bottom) of CaV1.2 (HEK293) recorded on the SyncroPatch® 96.

Cav3.2 - Online Analysis

SyncroCav32IV_OA

Shown are individual current voltage relationships of CaV3.2 (HEK293) obtained in a single run on the SyncroPatch®96. 75 % of the cells had a seal above 0.5 GΩ (color coded in green). The corresponding raw current traces of this experiment are shown in the data set CaV3.2 - Raw Currents.

Cells were kindly provided by Cytomyx Millipore, UK.

Cav3.2 - Raw Currents

SyncroCav32Currents

Shown are raw current responses of CaV3.2 (HEK293) to a current voltage relationship step protocol. 75 % of the cells had a seal above 0.5 GΩ (color coded in green). The corresponding online analysis of this experiment is shown in the data set CaV3.2 - Online Analysis.

Cells were kindly provided by Cytomyx Millipore, UK.

Epithelia Sodium Channel on the SP96

SP96 Enac Amiloride

The data present cumulative dose-dependent block of ENaC by Amiloride. Currents were obtained performing ramps -100mV/100mV every 2 s from a Vhold=-40mV. The large window shows the effect of increasing concentrations (1 µM, 10µM, 100µM) of Compound on the ENaC currents. The IC50 with Amiloride was 2.3 µM. Data were collected with a success rate of ~70 %.

Erythrocytes

Erys

Shown is a current recording from an erythrocytes as a response to a voltage ramp from -100 mV to +100 mV aquired on the SyncroPatch® 96. The increase in current was induced by a reduction in osmoliarity.

Cells were kindly provided by Dr. Andrea Brüggemann.

GABA a1b3g2 Antagonists

GABAa1_Antagonists

Pharmacology on GABAA α1β3γ2 as recorded on a multihole (4x) plate. Mean concentration response curves for Bicuculline, IC50 = 470 nM (n = 14); for a5IA IC50 = 461.19 pM (n = 9), maximum block was 29% at 100 nM; for FG7142 IC50= 54.52 nM (n = 15), maximum block was 58% at 10 μM; for MRK016 maximum current inhibition was 44.8% at 1 uM, IC50= 5.98 nM (n = 11). Cells were kindly provided by Millipore.

GABA a1b3g2 Bicuculline Dose Response

GABAa1_Bicuc_DR_vert

Pharmacology on GABAA α1β3γ2 as recorded on a multihole (4x) plate. Raw data traces of one exemplary recording using control solution (A) and increasing Bicuculline concentrations and a subsequent  washout (B). Cells were held at a constant holding potential of -70 mV and GABA was applied for approximately 2 s. After 3 control applications of 3 μM GABA, increasing concentrations of inhibitors were applied. Cells were preincubated with antagonists before co-applicaiton with GABA. Cells were kindly provided by Millipore.

GABA a1b3g2 Success Rates

Seal_Stat_GABAa1

Statistic of hGABAA a1b3g2 cells recorded on one NPC-96 1-hole (1x) patch clamp chip. Cslow = 18.8 ±1.6 (n=32), Rs = 8.6 ± 1.5 (n=32). 41.66 % of the cells on one NPC-96 chip (total n=96) had seal resistance > 1 GOhm at the beginning and at the end of experiment. 63.4 % of the cells had a seal resistance above 500 MOhm, which remained stable throughout the experiment.

Cells were kindly provided by Millipore.

GABA a5b3g2 Antagonists

GABAa5_Antagonists_small

Mean concentration response curves for Bicuculline, IC50 = 327 nM (n = 14); for a5IA IC50 = 933 pM (n = 11),  maximum block was 45% at 100 nM; for FG7142 IC50= 2.5 mM (n = 9), maximum block was 64.3% at 10 μM; for MRK016 maximum current inhibition was 52% at 1uM, IC50= 1.02 nM (n = 15).

Cells were kindly provided by Millipore.

GABA a5b3g2 Dose Response

GABAa5_ver

Pharmacology on GABAA α5β3γ2 as recorded on the SyncroPatch96. Raw data traces of one exemplary cell using increasing GABA concentrations (A) or increasing Bicuculline concentrations and a subsequent washout (B).

Cells were kindly provided by Millipore.

GABA a5b3g2 Success Rates

Bar_Seal_cm_Rs_GABAa5AppNote

Statistic of hGABAA α5β3γ2 cells recorded on one NPC-96 patch clamp chip. Cslow = 26.2 ±1.8 (n=32), Rs = 4.1 ± 0.2 (n=32). 53 % of the cells on one NPC-96 chip (total n=96) had seal resistance > 1 Giga Ohm at the beginning, 47 % at the end of experiment. 76 % of cells reached a seal resistance above 500 MΩ, which remained constant throughout the experiment.

Cells were kindly provided by Millipore.

GABA CRC Analysis

GABA on-line analysis results

Online analysis results of the dose response curves were obtained with a few mouse clicks. The color code of the online analysis helps to visualize different concentrations applied.
The corresponding data traces is shown in the data set "GABA Dose Response Curves".

GABA Dose Response Curves

SyncroPatch GABA CRC

Increasing concentrations of GABA were applied to each cell. Cells were from frozen stocks and were prepared for a FliPR experiment running alongside.
The corresponding online analysis of this experiment is shown in the data set "GABA CRC Analysis".

GABA Modulator: Diazepam

Diazepam

Shown is a raw current response of a HEK293 cell expressing GABAA receptors to initial exposure to GABA, followed by joint exposure to GABA and diazepam (as indicated). Solutions were  stacked (layered) in the pipette to achieve brief exposure times.

GABA On-set of response

Gaba-and-close-up_bea

With the SyncroPatch® 96 currents can be recorded continuously during compound application. This, in conjunction with a solution exchange time of about 100 ms, makes the SyncroPatch®96 an excellent tool for investigations of ligand gated ion channels. In the data example, 10 μM GABA was added to a HEK293 cell expressing GABAA (α1β2γ2) receptors. The boxed trace shows a close up of the GABA-response.

GABA Screening - User Interface

GABAa1_screenshot
Graphical user interface of the screening software used on the SyncroPatch96. Screenshot of depiction of raw data traces of hGABAA a1b3g2 expressing cells as recorded on one NPC-96 multihole (4x) patch clamp chip. 96 small color-coded pictures as seen in the upper left part display the individual recording wells. One highlighted experiment is displayed below, 16 other selected experiments are displayed on the right. Highlighted graphs show raw data current traces of the sum of 4 cells which were exposed to 3 μM GABA consecutively 7 times, including intermittent wash steps.
Cells were kindly provided by Millipore.

hERG Current Voltage Relationship

HergIV_No_and_with_Leak

Borosilicate glass chips are used as the patch clamp substrate, ensuring excellent voltage control of the cell membrane and high quality seals. Voltage gated channels such as hERG (expressed in HEK293) have been used to validate the system. These traces show the raw current responses of a single cell to a hERG IV pulse protocol. The data were recorded on the SyncroPatch® 96. In the upper screenshot raw current traces are shown. In the lower one the same current traces after leak subtraction are shown.

Cells were kindly provided by Cytomyx Millipore, UK.

hNav1.5 - 96-Well Plate Recording Format

Nav15_Screenshot

Shown is a screenshot made during a recording of hNaV1.5 currents stably expressed in HEK293 cells. On the top left the raw currents from each individual cell are displayed. The background color coding marks the cells with seals above 100 MΩ in blue and above 0.5 GΩ in green. On the right a selection of 16 recording channels can be displayed on a larger scale. The recording in a single well can be selected for detailed visual assessment.

Cells were kindly provided by Cytomyx Millipore, UK.

hNav1.5 - Screening Mode

SyncroPatch_NaTrace_small

Compound effect on hNav1.5  was investigated. A two-pulse protocol (-90 mV / -130 mV) was used to establish if the different compounds blocked the Nav1.5 currents in a state-dependent manner.
The image shows raw data traces. Success rate for the experiment was 71% (>1 GOhm seal resistance), indicated by the green color.
The corresponding online analysis of this experiment is shown in the data set NaV1.5 - Screening Online Analysis.

Cells were kindly provided by Millipore.

hNav1.5 - Screening Online Analysis

Nav1.5 screening

The image shows the state dependent block at -130 mV (light blue) and -90 mV (dark blue) by the addition of increasing compound concentrations to the different recording wells. The large window shows the effect of increasing concentrations of Lidocaine (red box) on the Nav1.5 currents.
Cells were kindly provided by Millipore.

hNav1.5 Current Voltage Relationship

NaStrom_IV

Borosilicate glass chips are used as the patch clamp substrate, ensuring excellent voltage clamp of the cell membrane and high quality seals. Voltage gated channels such as hNaV1.5 (HEK293) have been used to validate the system. This data example shows the I/V characteristics and the corresponding raw current traces of a single cell from a recording on the SyncroPatch® 96.

Cells were kindly provided by Millipore.

hNav1.5 Inactivation Protocol

NaInactivation

Shown are raw current responses of HEK293 cells expressing hNaV1.5 to a double (inactivation) pulse protocol.

Cells were kindly provided by Cytomyx Millipore, UK.

hNav1.5 Lidocaine Dose Response

NaLidocaine

Timecourse of the NaV1.5 peak currents in response to exposure to different lidocaine concentrations (0 μM, 1 μM, 10 μM, 100 μM, 0 μM). Time points at which the external solution was exchanged is marked by the red lines.

Cells were kindly provided by Cytomyx Millipore, UK.

Internal Perfusion - Kv1.3

p41_1_IntPerf

The SyncroPatch® 96 supports internal perfusion allowing internal administration of compounds, second messengers and metabolites. Here, KV1.3 currents, endogenously expressed in Jurkat cells, were blocked by the internal administration of Cs+ followed by washout with Cs+-free internal solution.

Kv1.3 - 96-Well Plate Recording Format

SyncroPatch 96 Kv1.3

Shown is a screenshot made during a recording of KV1.3 currents stably expressed in CHO cells. On the top left the raw currents from each individual cell are displayed. The background color coding marks the cells with seals above 500 MΩ in blue and above 1 GΩ in green.

Cells were kindly provided by Evotec AG, Hamburg, Germany.

Nav1.5 inhibition by Lidocaine

0NavLidocaineScreen

Inhibition of Nav1.5 currents in stem cell-derived cardiomyocytes (iCells) by lidocaine.  Lidocaine concentrations: 6 µM, 62 µM and 620 µM. The obatined  IC50 -value was 14 µM.

Cells were kindly supplied by CDI.

Nav1.7 Success Rate & Access Resistance

SuccessRate Nav17 Rs Cm Syncro

A Success rate (seal resistance) of ND7-23 cells on the SyncroPatch 96. Shown is a bar graph of seal resistances on the SyncroPatch®96 at the start (blue) and end (grey) of the experiment. B Bar graph of cell capacitance (Cslow) of ND7-23 cells. Mean Cslow = 19.9 ± 0.8 pF (n = 75 ). C Bar graph of series resistance (Rs) values for ND7-23 cells on the SyncroPatch 96. Mean Rs = 9.1 ± 1.3 MΩ (n = 75). The cells were kindly supplied by Millipore.

 

Nav1.8 Block by Lidocaine

Lido_traces_ConcResponse

A Raw traces from an exemplar cell recorded on the SyncroPatch 96 showing inhibition of current by increasing concentrations of lidocaine. Shown are current responses to a single step protocol to 20 mV for 25 ms from a holding potential of -120 mV. B Average concentration response curve for lidocaine, IC50 = 178 ± 11 μM (n = 35).

Cells were kindly provided by Millipore.

Nav1.8 Block by Tetracaine

Tet_traces_Conc_response

A Raw traces from an exemplar cell recorded on the SyncroPatch®96 showing inhibition of current by increasing concentrations of tetracaine. Shown are current responses to a single step protocol to 20 mV for 25 ms from a holding potential of -120 mV. B Average concentration response curve for tetracaine, IC50 = 71 ± 5 μM (n = 40).

Cells were kindly provided by Millipore.

Nav1.8 I/V Characteristics

ND723_Nav18_Syncro_IV_Figure

A Raw traces from an exemplar cell recorded on the SyncroPatch 96. Shown are current responses to increasing voltage steps from -60 to +60 mV. B Average current-voltage plot, Vhalf of activation was 12 mV (n = 32). C Average inactivation plot, Vhalf of inactivation was -27 mV (n = 32). Nav1.8 currents started to activate at about -30 mV, peak response was elicited between 20 and 30 mV and Vhalf of activation was 12 mV. The Vhalf of inactivation was -27 mV in good agreement with the literature. The ND7-23 cells were kindly provided by Millipore.

Nav1.8 Success Rate & Access Resistance

SuccessRate Seal cm Rs ND723 Syncro

A Success rate (seal resistance) of ND7-23 cells on the SyncroPatch 96. Shown is a bar graph of seal resistances on the SyncroPatch®96 at the start (blue) and end (grey) of the experiment. B Bar graph of cell capacitance (Cslow) of ND7-23 cells. Mean Cslow = 22.6 ± 0.8 pF (n = 88 ). C Bar graph of series resistance (Rs) values for ND7-23 cells on the SyncroPatch 96.Mean Rs = 7.1 ± 0.4 MΩ (n = 88). The cells were kindly supplied by Millipore.

Nicotinic Acetylcholine Receptors a7

SyncronAChR

Human nicotinic ACh receptors were activated by 100 µM ACh. Solution switch time was fast enough to see nAChR a7 responses.


P2X2/3

P2X23_10uATP

Shown are three consecutive current responses of 1321 N1 cells expressing P2X2/3 receptors to 10 µM ATP. Cells were washed twice between compound applications (holding −80 mV). The data demonstrate the reproducability of the whole cell current responses.

Cells were kindly supplied by Evotec AG, Hamburg, Germany

PNU modulation of nAChR a7

PNU_Syncro

After the desensitization of the nAChR a7 receptor by 100 µM acetylcholine, 10 µM PNU-120596 was applied together with 100 µM of Ach, reactivating the ion channels.

Cells were kindly provided by Galantos Pharma GmbH.

Seal Statistics

Seal Stats

These statistics summarize experiments conducted with HEK293, CHO, Ltk, RBL, and Jurkat cells. More than 50 % of the cells have a seal resistance above 1 GΩ. In total more than 80 % of the cells had a whole cell resistance above 0.5 GΩ.


Stabe GABA Responses on Repeated Compound Addition

Repeated GABA responses

The traces show repeated application of 100 µM GABA to GABAA receptors (α1 β2 γ2) expressed in HEK 293 cells. Current amplitudes were stable over time, illustrating the reliable and fast solution excahnge supported by the SyncroPatch 96.

TRPA1

Activation with AITC and block with HC030031

Screenshot of online analysis data of hTRPA1 expressing HEK 293 cells as recorded on one 4x hole NPC-96 patch clamp chip. Currents were obained by performing ramps from -50 mV to +50 mV. Graphs show the summation of the current amplitudes of 4 cells per well, which were activated by 3 μM AITC and inhibited by 30 μM HC030031 plotted against time. For highlighted experiments, note the blue shade overlaid on the curves represents the full block with HC. The grey shade represents the activation step, the white color the wash steps. Cells were kindly provided by EMD Milipore.

TRPV1

TRPV1_bea_verzerrt

Using the SyncroPatch® 96 CHO cells expressing TRPV1 were subjected to a voltage ramp (-100 mV to +100 mV) in the presence and absence (control) of 2 μM capsaicin. As shown in the exemplar traces, inward and outward currents increase upon capsaicin addition.