Application Note

Channel:Cells:
Tools:

hNaV1.5
CHO
SyncroPatch 384PE
SyncroPatch 384i

Increase throughput by recording inunattended mode on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by Charles River.

Summarythe

For efficient experiments, especially in unattended

pharmaceutical industry to aid drug discovery. Largethroughput

screening

(HTS)

is

used

in

mode, success rates must be high. On the SyncroPatch

numbers of chemical compounds can be tested for

384, cells, solutions, compounds, and 10 x NPC-384 chips

biological activity using a range of techniques. The

were loaded into position on the robot and the experi-

patch clamp technique remains the gold standard toments were run unattended for 6 hours. First of all, com-test activity of compounds on ion channels and auto-

pound plates were prepared for all the experiments,

mated patch clamp (APC) is increasingly adopted infollowing this, the gripper arm moved a new NPC-384

HTS labs as an alternative to conventional patch clamp

chip and compound plate automatically into position

given its increased ease-of-use and higher through-when the previous experiment was finished. Alternatively,

put. APC is employed in all aspects of drug discovery

compound plates can be prepared between runs orfrom hit finding and lead optimization through to

pre-prepared compound plates can be stacked in thetarget validation and safety testing. This is only possible

robot and the on-board barcode scanner can be used todue to the increase in throughput toward HTS capabilities,

document the compound plate used. Sufficient cells andthe compatibility with HTS workflows, and a lower cost per

solutions were loaded into position for the unattended

data point which can compete with other techniques

period, including the use of four refillable reservoirs forsuch as fluorescence imaging (using, for example, theinternal, external and wash solutions. Success rates for

FLIPR™ instrument) and calcium imaging with the add-

completed experiments were 94-97% with little change

ed benefit of real-time kinetics of drug effects. Indeed,in the success rate for the last (10th) plate (94%) at the

all the major contract research organizations worldwide

end of the unattended period compared with the firstuse APC for ion channel screening and cardiac safety

plate (97%) at the start. The success rates for completed

testing. Increased automation, including unattended
operation, is also an important factor for increasing
throughput, and instruments can reliably work beyond
an 8-h day provided they are serviced with enough cells,
solutions, and compounds1. For this to work effectively, data
must be reliable with high success rates, low false positive
and negative rates along with reproducible IC50 values.
Here,

we

demonstraterecordingsofhNaV1.5

expressed in CHO cells on the SyncroPatch 384 in unattended mode. In a 6 hour period, 10 compound plates
were prepared, 10 NPC-384 chips were recorded andsuccess rates, IC50s and Z’ values were calculatedusing 7 different compounds in duplicate on eachplate, along with positive and negative controls.
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experiments for all 10 chips is shown in Figure 1.
100
94
Success Rate (%)

High

Results

50

0

n01 n02 n03 n04 n05 n06 n07 n08 n09 n10
Chip number

Figure 1: Success rate for completed experiments for 10 NPC-384 chips.
Success rate (% of 384 wells used for analysis) of 10 NPC-384 chips is shown
for recordings over a 6 hour period in unattended mode.

Application Note

Along with high success rates, data must also be reliable% changewith low false positive and negative rates and a Z’ value
close to 1. The Z’ value is a statistical parameter used inZ’ value

Chip #

Peak 1

Peak 2

Peak 1

Peak 2Chip 1 (40)5.5 ± 0.9 (1)10.9 ± 1.1 (4)

0.91

0.89

Chip 2 (36)4.1 ± 1.2 (0)4.9 ± 1.3 (0)

0.90

0.89

Chip 3 (35)6.3 ± 0.9 (0)6.8 ± 1.0 (0)

0.91

0.88

Chip 4 (38)1.8 ± 0.8 (0)2.4 ± 0.9 (0)

0.91

0.90

Chip 5 (37)3.8 ± 1.1 (0)4.3 ± 1.3 (1)

0.89

0.88

Chip 6 (36)3.1 ± 0.9 (0)3.6 ± 1.0 (0)

0.92

0.91

Chip 7 (38)5.7 ± 0.8 (0)5.4 ± 0.9 (0)

0.91

0.91

(50 µM tetracaine) for the 10 chips (Shown in Table 1).

Chip 8 (40)5.2 ± 0.7 (0)4.7 ± 0.8 (0)

0.91

0.90

Rundown was low which is shown in the scatter plot in

Chip 9 (40)3.0 ± 0.7(0)1.8 ± 0.8 (0)

0.93

0.93

Figure 2 for Peak 1 and Peak 2 and in Table 1. In 380 wells

Chip 10 (40)5.3 ± 1.2 (2)9.7 ± 1.4 (3)

0.88

0.87

over the 10 plates which were used for negative control,

Mean

4.4 ± 0.3

5.4 ± 0.9

0.908 ± 0.004

0.893 ± 0.006

HTS (and elsewhere) to evaluate the overall quality ofan assay without intervention of test compounds2,3, it iscalculated using positive and negative control data.
Using the double step protocol shown in Figure 2A,
Z’ values were calculated for the 1st and 2nd peak fornegative control (0.3% DMSO) and positive control

just 3 wells for Peak 1 and 8 wells for Peak 2 showed adecrease in current >20% compared with reference
giving a low false positive rate of 0.8% for Peak 1 and 2.1%
for Peak 2. There were no wells which showed an increasein current >20% compared with reference giving a false
negative rate of 0%.
A peak 1
peak 2
1s-15 mV-15 mV, 20 ms

2 nA

During the unattended run, 7 compounds were applied in
duplicate to each chip (see Methods). In addition to this,10 mspositive (8 wells; 50 µM tetracaine) and negative controls (40Peak 1

wells; 0.3% DMSO) were performed on each plate. Figure 3

80

shows 16 concentration response curves for Peak 2 for an

60

example compound (clozapine) overlaid. The concen-

40

tration response curves were in excellent agreement

20

across the plates, and could be almost exactly overlaid.

0

1.0
10

9

p

hi
C

hi

hi
C

p

8

hi
C

C

p

6
hi
p

7

5
hi
p

C

hi

p

4
p

3
p
hi

C

C

100

C

C

C

hi

hi

p

p

1

2

-20

Peak 2% change in current

80

60

Peak 20.8
Normalized block% change in current

peak 2

not displayed-100 mV

B 100

peak 1

Table 1: Negative control and Z’ values for 10 NPC-384 chips for Peak 1
and Peak 2. Chip number is shown along with number of wells used
for negative control in brackets. Mean ± S.E.M of % change in current
for Peak 1 and Peak 2 and the number of wells which displayed >20%
change in current given in brackets. Z’ values were calculated from thenegative and positive control data for each plate, mean ± S.E.M is also
shown for Peak 1 and Peak 2. Z’ are between 0.5 and 1 indicating an
excellent assay2,3.

0.6

0.4

40
20
0

10
hi

p

9
p
hi
C

C

8
hi
C

p

hi
C

p

7

6
hi

C

p

5
hi

C

p

4
p
hi
C

hi

p

3

2
C

C

hi

p

1
p
hi
C

n03 IC50 2

n07 IC50 2
n08 IC50 1
n08 IC50 2

n05 IC50 2

n09 IC50 2

n07 IC50 1

n04 IC50 1
n04 IC50 2
n05 IC50 1-20

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n06 IC50 1
n06 IC50 2

n03 IC50 1

0.2

Figure 2: Low rundown of current responses. A. The voltage protocol
used for the experiments is shown (left) along with an example of thecurrent traces from a cell (right) showing also the cursors used for analysis.
B Change in current (%) in negative control (0.3% DMSO) solution is shownfor Peak 1 (top) and Peak 2 (bottom) for all chips. % change is shownnormalized to reference at the beginning of the recording on each chip.
Each point represents 1 well. Dotted lines indicate change of ±20%n02 IC50 1
n02 IC50 2

0.0
10-7

2

3 4 562

3 4 5610-5
10-6[Clozapine (M)]2

3 4 56n09 IC50 1

10-4

2

3 4 56

Figure 3: Concentration response curves for Peak 2 from 8 plates forclozapine overlaid. Concentration response curves are very similar acrossthe plates.

Application Note
A Chip 7 - Peak 1

B

4
3

Chip 7 - Peak 2

R2 = 0.975

Log IC50 (μM) value 2

2

10
7
6
5
4
3
2

1
7
6
5
8 9

2

1

4

3

5

6

7

8 9

10
Log IC50 (μM) value 1

2

3

4

Figure 4: Heat maps of compound effects and IC50 correlation on the SyncroPatch 384. A Top left: Heat map of Chip 7 Peak 1 - effect of compounds,
negative control (wells H1-20 and I1-20) and positive control (wells H21-24 and I21-24). Top right: Heat map of Chip 7 Peak 2 - effect of compounds, negative
control (wells H1-20 and I1-20) and positive control (wells H21-24 and I21-24). Color code is shown on the top right. B Correlation plot of IC50 values. On thecompound plates, the concentration response curves for each compound were run in duplicate on each plate. The two IC50 values for each chip for Peak 1
and Peak 2 are plotted against each other and fit with the Pearson’s correlation coefficient. R2 = 0.975 indicating excellent correlation.
• Automatic unload of chip
• Movement of compound plate into
correct position
End of user input

5 min• Check protocol• Load NPC-384 chips
etc.
• Press start

User input

3 min~30 min~ 1 hrCompound preparation (10individual compound plates)

Measurement of 1 NPC-384 chip:• Cell capture & sealing• Control recordings & QC
• Compound addition

5 min

Offline• Unload finalchip• Wash andshutdown

Analysis

Figure 5: Timeline of unattended operation on the SyncroPatch 384 for the hNaV1.5 assay. Chips, solutions and compound stock solutions were pre-loaded
into the robot. The robot first prepared all the compound plates for the 10 experiments which took approx. 1 hour. Following this, one experiment, fromchip load and priming, through cell capture & sealing, IV and QC, control recording and a single addition of compound to each well, took approximately
30 mins. At this point, the chip was unloaded and a new compound plate was added to the correct position, a new chip loaded and the experimentrestarted. In a 5 hour measurement period, 10 x NPC-384 chips were recorded. After loading NPC-384 chips, compounds and solutions, no further user input
was required until after the 6 hour unattended period. Data analysis was performed offline after completion of the experiments. Note that experiment
time for other assays/targets will be different depending on whether single or cumulative addition of compound concentrations is performed, or where
extended incubation times are required (e.g. for hERG experiments), and compound plates can be prepared prior to each NPC-384 chip recording rather
than all at the start of the experiment, depending on user preference.

Download more Application Notes from www.nanion.deApplication Note
Compound

IC50 (µM)

Amitriptyline

IC50 (µM) Literature

Peak 1

Peak 2

Ref. 4
Kramer(Peak 1)5.4 ± 0.3

0.67 ± 0.03-

Ref. 5
Crumb(Peak 1)
5.8

Bepridil

7.6 ± 0.3

1.73 ± 0.06

2.3 ± 0.3

2.9

Chlorpromazine

4.7 ± 0.1

1.14 ± 0.03

3.0 ± 0.4

4.5

Clozapine

35.4 ± 0.6

8.1 ± 0.2

15.1 ± 2.7-

Flecainide

6.7 ± 0.2

5.5 ± 0.1

6.2 ± 0.6

6.7

Quinidine

36.3 ± 1.4

25.6 ± 0.8

14.6 ± 1.0>5.4

Verapamil

26.1 ± 0.7

8.4 ± 0.3

32.5 ± 4.2>1.0

Table 2: Average IC50 values for 7 compounds on NaV1.5 Peak 1 andPeak 2 recorded in unattended mode on the SyncroPatch 384. IC50 values are mean ± S.E.M of 16 experiments. Values are also compared withthe literature from Refs. 4 & 5, equivalent of Peak 1, where possible, or
highest concentration is given (quinidine and verapamil, ref. 5).

Figure 4 shows the heat map of an example chip. For
some compounds, Peak 2 is blocked with higher potency
and this can be observed easily on the heat map (colour
code is shown in the right of Figure 4). Figure 4B shows thecorrelation plot of all 7 compounds where the IC50 fromeach chip is plotted against its duplicate counterpart forPeak 1 and Peak 2 and Table 2 shows the average IC50

values for all compounds tested with comparison to the literature. All IC50s recorded agreed well with the literature4,5.

References

In conclusion, the SyncroPatch 384 instruments (SyncroPatch 384PE and SyncroPatch 384i) can reliably run in
unattended mode as described in this Application Note.
We chose hNaV1.5 and a double voltage step protocol toinvestigate potency of compounds on the resting(Peak 1) and inactivated state (Peak 2) of the receptor. In a 6 hour unattended period, compound plates
were prepared by the robot followed by recording of
10 NPC-384 chips. Seven compounds were used oneach plate, along with positive and negative controls.
A single concentration of the compound was added
to the wells, with 6 replicates for each concentration.
The concentration response curves were constructedacross multiple wells (4 point). This was performed in
duplicate on each plate on both Peak 1 and Peak 2
giving rise to 224 (4 point) concentration response curves.
Data was reliable with high success rates (>94%) for completed experiments, low rundown or run-up, therefore
low false positive and negligible false negative rates,
excellent Z’ values and concentration response curves
with IC50 values in good agreement within plates, acrossplates and with the literature4,5. Therefore, users of theSyncroPatch 384 instruments can confidently extend
their workday and increase productivity by using the
instruments in unattended mode.
Electrophysiology
Perforated patch clamp recordings were conducted

1. Obergrussberger, A., et al. 2020. EODD. doi: 10.1080/

according to Nanion’s standard procedure for the17460441.2020.1791079

SyncroPatch 384 instruments using multihole chips (4x2. Zhang, J-H., et al. 1999. JBS. 4 (2): 67-73

holes per well). Cells were held at -100 mV. A voltage

3. Iversen, P.W., et al. 2006. JBS. 11 (3): 247-252

step to -15 mV for 1 s for Peak 1 was followed by a brief

4. Kramer, J., et al. 2013. Sci. Rep. 2013. 3: 2100

step back to -100 mV for 40 ms and then a step to -15 mV

5. Crumb, W.J. Jr., et al. 2016. JPTM. 81:251-62

for 20 ms for Peak 2. This double step protocol was
repeated every 10 s. Single concentration addition was

Methods

performed

andconcentration

response

curvescalculated across multiple wells using DataControl 384.
Cells

The same concentration of compound was added to 6

CHO cells expressing hNaV1.5 were kindly provided bywells (i.e. 6 replicates) and 4 concentrations for eachCharles River.

compound were used. Compounds were added in
duplicate to chips 2-8, chips 1 (start of experiment) & 10

Cell culture(end of experiment) received only negative control (0.3%

Cells were cultured and harvested according to

DMSO) to check for rundown, with 8 wells receiving

Nanion’s standard cell culture protocol.

positive control (50 µM tetracaine). Analysis was
performed offline using DataControl 384, Excel and
IgorPro (Wavemetrics).

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