Application Note

Channel:Cells:
Tools:

NaV1.5 and hERG
CHO and HEK293
SyncroPatch 384

There is no F in APC: Reliable fluoride-freerecordings on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly supplied by SB Drug Discovery or Charles River.

SummaryResults

Automated patch clamp (APC) instruments are usedhERG expressed in HEK293 cells were recorded on thefor a wide variety of applications ranging from basic

SyncroPatch 384 using fluoride-free (FF) internal solution

researchbiophysical(Figure 1A) or standard (fluoride-containing) internal solu-

characteristics of ion channels, through to routine

tion (Figure 1B). Little difference in current amplitude (Fig-cardiac safety testing. Their use in cardiac safetyure 1C) and RSeal (Figure 1D) was observed between theintochannelopathies

andscreening has increased over the years and APC is
now an established and accepted technique in most,two groups (Figure 1C).
Control at start
AB

Control at end
Full block (E4031)

if not all, safety testing laboratories. It is well known thatfluoride is often used in the internal solution in APC

Control at start
Control at end
Full block (terfenadine)

experiments to improve the seal resistance. The presenceof external calcium (or other divalent cation) further
improves the seal by a mechanism thought to be due tothe formation of CaF2 crystals at the interface between
the pipette or micro-pore and the cell as described in aDC
2

1and allows stable measurements to be performed over(182)long periods of time8. However, because there are someinternaltousesolutions

andexternal solution that does not use divalent ‘sealenhancer’ solutions, we have developed a method thatallows fluoride-free, physiological solutions to be usedwith good success rates. We demonstrate this using thecardiac ion channels hERG expressed in HEK293 cells(SB Drug Discovery) and NaV1.5 expressed in CHO cells(Charles River).

Download more Application Notes from www.nanion.de

1.00.500.0st
in an
te da
rn rd
al

fluoride-freeadvantageous

1.5

al

isnt
rol
st
co an
nt da
ro rd
FFl
Full blockst
Fu an
ll b dalo rdck

physiological,itcowhereFF

experiments(152)

2.0

rn

phosphatase7. Fluoride is used because it improves the seal

2.5

(152)

testeady-state fast inactivation4-6 and its inhibition of protein(182)

200 msineffects on voltage dependence of conductance, andImax (nA)gated Na+ channels for over 20 years2,3,4, despite known

RSeal (GΩ)

experiments, fluoride has been used to record voltage-0.2 nA

FF

recent patent application . Even in manual patch clamp
1

Figure 1: hERG-mediated currents recorded on the SyncroPatch 384.
A hERG currents from an example cell recorded using FF internal solution.
B hERG currents recorded from an example cell using standard internalsolution. C Box plot of current amplitudes for an example NPC-384 chipin FF and standard internal solution. The distribution of current amplitudes
is not statistically significantly different (P=0.7, unpaired Student’s t test).
The current amplitude after block with E4031 or terfenadine is also shown.
D Bar graph of RSeal values for one experiment for 182 and 152 wells for FF
and standard internal, respectively. Shown are mean ± S.E.M.

Application Notethe start of the experiment in either FF or standard internal.
The hERG current was completely inhibited by a full block
concentration of either E4031 or terfenadine (Figure 1).
Success rates (% available wells) were calculated for

100

100

80

80

standard internal (n = 15)
fluoride-free internal (n = 17)

Available wells (%)

after 5 min was not statistically different from the current at

B

Available wells (%)

Current amplitude was also stable over time, the current

A60

60

4040

multiple NPC-384 chips using either standard internalsolution or FF internal solution and no use of ‘seal enhancer’.

20

2000end of the experiment was used, giving a success rate ofalmost 60% for completed experiments. There was little lossof wells over time, the success rate at the end of the experiment was similar to that at the start (Figure 2).

A

B

100

100

standard internal (n = 10)
fluoride-free internal (n = 32)

eR ndSeoal≥ fe
0. xp
25 .
G
Ω

9% if the quality control parameter RSeal ≥ 0.25 GΩ at thestat the end of the experiment, and this increased to 57 ±R artSeoal≥ fe0. xp
25 .
GΩ

R art
Seoal
≥ fe
1 xp
G .
Ωwere achieved without fluoride for wells with RSeal ≥ 1GΩ

stused, as expected, success rates of 34 ± 8% (n = 32 chips)eR ndSeoal
≥ fe
1 xp
G .
ΩAlthough success rates were better when fluoride wasFigure 3: Bar chart of success rates for experiments using standard internal
solution and FF solution for CHO cells expressing NaV1.5. A Bar chart using
QC parameter RSeal ≥ 1 GΩ at the start and end of the experiment. B Barchart using QC parameter RSeal ≥ 0.25 GΩ (usable wells) at the start and endof the experiment. Shown are mean ± S.D. values for 10 (standard internal)and 32 (FF) NPC-384 chips.

NaV1.5-mediated currents were stable over time in bothFF physiological internal solution or standard internal solu-tion (Figure 4). Peak amplitudes were, however, larger in
FF internal solution compared with standard internal solu-

80

Available wells (%)

80

60

60

4040

2020tion (p<0.01, unpaired Student’s t test; Figure 3C). NaV1.5

was completely inhibited by tetracaine (full block).

AB
Control at start
Control at end
Full block(tetracaine)
0.5 nA
10 ms

0.25 GΩ was used and 86 ± 6% for standard solution (Fig-ure 4B), a cutoff which indicates an acceptable RSeal forscreening. There was little loss of wells over time, the suc-cess rate at the end of the experiment was similar as atthe start.

Download more Application Notes from www.nanion.deRSeal (GΩ)

(132)

1.5
1.0-1

0.5

-2

0.0l

ure 3A) were achieved. This increased to a success rateof 55 ± 12% for FF if the quality control parameter RSeal ≥ntrol
stco annt daro rdl
FFFull bst loc
Fu an kll b dalo rdckco(121)2.0nawere 42 ± 9% in FF versus 78 ± 5% in standard solution (Fig-0-3(121)(244)

interchannel. Using these cells, success rates for RSeal ≥ 1 GΩ

2.5

FFWe also used CHO cells expressing the cardiac NaV1.5

FFFigure 2: Bar chart of success rates for experiments using standard internal
solution and FF solution for HEK293 cells expressing hERG. A Bar chart using
QC parameter RSeal ≥ 1 GΩ at the start and end of the experiment. B Barchart using QC parameter RSeal ≥ 0.25 GΩ (acceptable RSeal for screening) atthe start and end of the experiment. Shown are mean ± S.D. values for 10(standard internal) and 32 (FF) NPC-384 chips.

DCImax (nA)eR ndSeoal
≥ fe
0. xp
25 .
G
Ω

st
R art
Seoal
≥ fe
0. xp
25 .
G
Ω

e
R ndSeoal
≥ fe
1 xp
G .
Ωst

R art
Seoal
≥ fe
1 xp
G .
Ω0stin ante darn rdal

0Figure 4: NaV1.5-mediated currents recorded on the SyncroPatch 384.
A NaV1.5 currents recorded from an example cell using FF internal solution.
B NaV1.5 currents recorded from an example cell using standard internal
solution. C Box plot of current amplitudes for an example NPC-384 platein FF and standard internal solution. The current amplitude after block withtetracaine is also shown. D Bar graph of RSeal values for one experiment for
121 and 244 wells for FF and standard internal, respectively. Shown aremean ± S.E.M.

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of raw current tracesof hERG expressed in HEK293 cells using FF physiological internal solution. Three hundred and eighty-four small color-coded pictures as seen in the upper leftpart display 384 recordings. Wells are color-coordinated based on RSeal where green wells have RSeal > 500 MΩ, blue wells have RSeal between 100 and 500
MΩ and light-blue or grey wells are < 100 MΩ or disabled. In this case, over 61% of wells had RSeal > 500 MΩ.

ACB

3RSeal (GΩ)

Control at start
Control at end
Full block(tetracaine)
0.5 nA

0.5 nA
10 ms

4

10 ms2

1stin ante darn rdal

FFinternal

0Figure 6: NaV1.5 at physiological temperature in FF and standard internal. A NaV1.5 currents recorded from an example cell using FF internal solution at
physiological temperature B NaV1.5 currents recorded from an example cell using standard internal solution at physiological temperature. C Bar graph ofRSeal values for one experiment for 194 (FF) and 357 (standard) wells. Shown are mean ± S.E.M.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384wells) in FF and 2.98 ± 0.01 GΩ (n = 357 wells) in standard

software during an experiment. A color-coded over-internal (Figure 6). NaV1.5-mediated currents were also

view (based on seal resistance in this case) of all 384

stable over time (Figure 6).

wells gives the user a good impression of the successrate of the experiment. The user can choose whether toThe SyncroPatch 384 is a high throughput and highlyvisualize raw traces or online analysis. In this case,reliable automated patch clamp device which can

raw current traces are shown. Over 60% of thebe used with standard (fluoride-containing) internalwells had RSeal > 500 MΩ and, therefore, wells which

solutionwere used for analysis, in FF physiological internal

recordings.

solution. With our new FF application, the user has theinternal solution (and no external seal enhancer) can

opportunity to run FF physiological solution with nobe used with good success rates, making it possible toexternal seal enhancer. Alternatively, internal fluoride

perform high throughput APC recordings in physiologicalcan be used to achieve success rates >80% for com-solutions used in manual patch clamp. Taking advan-

pleted experiments with our standard approach. Fur-tage of the 32-well mode of the SyncroPatch 384, wherethermore, we performed recordings with NaV1.5 at physi-part of the chip can be used at a time and the remainder

ological temperature (36 ± 1°C) where we achievedof the chip can be used at a later timepoint, increases

36 ± 9% (n=17; RSeal > 0.25 GΩ) at the end of the experi-

flexibility of the system making it an ideal system notfor

highsuccess

Alternatively,

rates

and

fluoride-freelong-lasting
physiologicalment without fluoride versus 89 +/-5 % (n=15; RSeal > 0.25

only for high throughput screening of large compoundGΩ) at the end of the experiment when internal fluoride

libraries or safety testing, but also for small screening

was used. The mean RSeal was 2.37 ± 0.01 GΩ (n = 194

projects and assay development.

References

Cell culture
Cells were cultured and harvested according to1. Løjkner, L.D. 2019. US Patent Application ‘Seal Enhancer’ Patent Number: 16/465590 (accessed 31.01.2022)
2. Cummins T.R., et al. 1999. J. Neurosci. 19(24): RC43. doi:
10.1523/JNEUROSCI.19-24-j0001.1999.
3. Renganathan M., et al. 2000. J. Neurophysiol. 84(2):710718. doi: 10.1152/jn.2000.84.2.710.
4. Rugiero F., et al. 2003. J. Neurosci. 23(7):2715–2725.
5. Jarecki B.W., et al. 2008. J. Physiol. 586.17: 4137–4153.
6. Coste B., et al. 2004. Mol. Cell. Neurosci. 26: 123–134.
7. Khatra B.S., et al. 1978. Biochem. Biophys. Res. Comms.
85(2): 647-654
8. Föhr K.J., et al. 2021. Front. Pharmacol. 12: Article
622489 doi: 10.3389/fphar.2021.622489

Nanion’s standard cell culture protocols.
Consumables
For Fluoride-Free recordings the recently developed
NPC-384FF chip was used, whereas for recordings withinternal fluoride, the standard NPC-384 chips were used.
Electrophysiology
Whole-cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch 384. hERG was recorded using a doublestep voltage protocol from a holding potential of -80 mVto +60 mV for 500 ms followed by a step to -40 mV for 500
ms repeated every 15 s. Peak amplitude was measured
at the start of the second voltage step. NaV1.5 was

Methods

measured using a single step protocol from -100 mV toCells

contained 110 mM KF, fluoride-free internal solution-10 mV repeated every 10 s. Standard internal solution

HEK293 cells expressing hERG were kindly supplied by SB
Drug Discovery. CHO cells expressing NaV1.5 (Cat:CT6007)

werekindly

suppliedbyCharlescontained110mM

K-gluconate.

Externalsolution

contained 60 mM NMDG. Please ask for more details.

RiverLaboratories.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960