Manual Patch Clamp Quality + Automated Patch Clamp = O

Manual patch claMp quality + autoMated patch claMp = O. Scheel P. van Stiphout tM cytopatch instruMent T. Knott

introduction The most important requirement in drug fer or compound over several minutes. discovery is the reliability of data. In Furthermore, due to the flexible perfu- terms of ion channel screening, the ma- sion system, the fast application of ago- nual patch clamp technique fulfils this in nists, as required for the investigation of an excellent way. However, it has a very fast desensitizing ligand ion channels, low throughput and high costs per data is possible with the CytoPatch™ Instru- point. The automated patch clamp offers ment. For example, the fast rise time cytocentrics Bioscience GMBh, Germany a much higher throughput and lower co- (10 - 90 %) for the nicotinic acetylcholi- address Joachim-Jungius-Str. 9 | D-18059 Rostock sts. But the data quality and the flexibility ne receptor Alpha 7, of less than 5 ms, of the perfusion schemes of the manu- is shown. In summary, the CytoPatch™ phone +49 (0)381 440 388 0 | Fax +49 (0)381 440 388 47 al patch clamp could not be complete- Instrument offers the same flexibility Mail [email protected] | weB www.cytocentrics.com ly reproduced. Cytocentrics addresses and data quality known from the ma- this demand with the CytoPatch™ In- nual patch clamp. Simultaneously, it strument. Its unique and patented Cyto- offers a higher throughput, lower co- centering Technology allows true giga- sts and a higher standardisation of the seal recordings without the need of seal measurement processes. Thus, it is an driving chemicals. Two sophisticated instrument that satisfies the contem- liquid-handling systems enable a non- porary needs and expectations on the stop perfusion of the cell with either buf- patch clamp technique.

BeneFit oF the cytopatchtM technoloGy coMparative herG screeninG TM The CytoPatch Instrument has a unique technique which is well suited in HEK293 Instant Cells for the investigation of ligand gated as well as voltage gated ion channels. herG study

Dofetilide E-4031 Terfenadine -3 Terfenadine +40 1.00 1.00 2 Quinidine R =0.93 CytoPatch CytoPatch tM -4 Metoprolol mpc mpc • Novel CytoCeNteriNg technoloGy enaBles true GiGaseals Astemizole 80- 0.75 0.75 h

c -5 Verapamil t a 200 pA t n

P Cisapride t e

• AvoidANCe of seAl-forCiNg buffers (No fluoride) o r n t r e

y -6 E-4031 u r r c C

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C . I -7 Bay_10 m r • PermANeNt buffer PerfusioN iN CoNtrol PhAse (

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o Bay_13 l n -8 Bay_14 0.25 0.25 • PermANeNt ComPouNd PerfusioN Bay_15 -9 Bay_17 IC 8.3 nM IC50 9.15 nM -9 -8 -7 -6 -5 -4 -3 50 Bay_12 0.00 0.00 • No Cross-CoNtAmiNAtioN of ComPouNd log (IC ) manual PC 50 Bay_18 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 • best results with „stiCky ComPouNds“ log (c/M) log (c/M) In a blinded study the effect of 18 compounds provided by Ba- Representative hERG outward currents recorded from Instant HEK 293 cells Concentration response relationship for the potent hERG inhibitors E-4031 (left) and terfenadine yer Schering Pharma (Wuppertal) investigated with the Cyto- stably expressing the hERG ion channel. Currents elicited by the voltage pro- (right) determined with the CytoPatch™ Instrument (grey symbols and curves). In comparison • seriAl resistANCe: 10 mohm Patch™ Instrument was compared to manual patch clamp re- tocol shown top right. data obtained with manual patch clamp recordings are depicted in red. cordings performed at the Bayer site (Assay Drug Dev Technol. • seriAl resistANCe ComPeNsAtioN √ 2011 Jun 15. (Epub ahead of print)). • low system CAPACity (< 10 Pf) • ultrA-fAst APPliCAtioN for lgiCs <10 ms • glP CoNform (hlPC ANAlysis √ ) • fully AutomAted: severAl hours wAlk-AwAy time tM Kv1.5 and Kv1.3 screeninG with the cytopatch instruMent • AutomAted dAtA-evAluAtioN

tM 3 the cytopatch instruMent provides the saMe hiGh data quali- 1.00 5 1.00 4-Aminopyridine 4-Aminopyridine 4 t t ty as the Manual patch claMp technoloGy. n 0.75 n 0.75 2 e e A A r r r n n r

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o HILLSLOPE -1.032 HILLSLOPE -1.169 n n 0 EC50 0.0001992 0 EC50 0.0002590 autoMated patch claMp with real Giga Ohm seals 0.00 0.00 0 100 200 300 400 500 -6 -5 -4 -3 -2 0 50 100 150 200 -6 -5 -4 -3 -2 time / ms log (c/M) time / ms log (c/M)

Conventional Patch clamping Whole-cell Kv1.5 currents stably expressed in CHO-K1 cells recorded with the CytoPatch™ Instrument (left). Whole-cell Kv1.3 currents stably expressed in CHO-K1 cells recorded with the CytoPatch™ Instrument (left). Concen- Concentration-response relationship for the inhibition of K 1.5 currents by the K channel blocker 4-AP (right). tration-response relationship for the inhibition of K 1.3 currents by the K channel blocker 4-AP (right). pressure suction recording v v glass pipette

adherent cell Fast liGand Gated ion channels cardiac sodiuM channel na 1.5 Automated CYTOCENTERING technique v

suspended nav1.5 currents recorded with i/v activation plot ic50 tetrodotoxin 500 ms EC Acetylcholine tM cell 50 the cytopatch instrument 1.00 Cyto 1.00 centering 0.5 500 pA e s 0.75

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pressure r suction i

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c Hillslope 1.304

c HILLSLOPE -1.028 -1500 Opening a -1.0

r EC50 1.053 mM EC50 3.375e-006

F 0.00 -2000 0.00 0 2 4 6 -5 -4 -3 -2 -1 -80 -55 -30 -5 20 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 time / ms log (c/M) Vm / mV log (c/M)

TM Single current trace of human nicotinic acetylcholine receptor alpha 7, stably expressed in CHO-K1 cells, measured Current voltage relationship for human NaV1.5 stably expressed in Hek 293 cells, measured with the CytoPatch In contrast to other planar patch clamp platforms the unique als and the whole-cell break through are obtained. The with the CytoPatchTM Instrument (left) and concentration response curve for the agonist acetylcholine measured with Instrument. Cells were clamped from a holding potential of –100 mV in 10 ms pulses to test voltages from –70 to the CytoPatchTM Instrument (right). +35 mV in 5 mV increments. Concentration-response relationship for the inhibition of Na 1.5 by Tetrodotoxin (right) CytoPatch™ Chip contains a real patch pipette that is sur- sealed cell is then continuously perfused with extracel- V rounded by the Cytocentering opening. The latter is used to lular buffer or test compound. This resembles the patch position the cell on the patch pipette. By application of pre- clamp process known from the manual patch clamp and cise pressure protocols through this patch pipette, gigase- results in the same high quality of recordings.