Round Table Discussion
Thoughts on APC assay optimization -
Strengths and weaknesses of APC instruments
Prof. Clemens Möll...
Correlation MPC-APC
Assay set up
Flexibility of operation;
primary cells
2
Round Table Discussion
Controlled
state of
channel
Low binding
of
hydrophobic
compounds
Low leak
currents
Control of
membrane
potential /
capacit...
Controlled
state of
channel
Low binding
of
hydrophobic
compounds
Low leak
currents
Control of
membrane
potential /
capacit...
Potential
(mV)
-100 -50 0
-2
-1
0
1
Peak current
(nA)
.
potential (mV)
-140 -120 -100 -80 -60 -40 -20 0 20 40
current(nA)
...
6
Excellent correlation between manual and planar patch clamp
Before employing automated (planar)
patch-clamping in our pr...
Key points for pharmacology: same as for MPC and HT systems
o Prepare compound solutions as freshly as possible. Observe s...
(Many) Port-A-Patch, PatchLiner and
SyncroPatch assays are easy to set up
o Operation in standard modes easy
o Very "forgi...
(Many) Port-A-Patch, PatchLiner and
SyncroPatch assays are easy to set up
o The healthier the cells are, the better the se...
APC instruments complement each other
In addition, the Port-A-Patch proved very useful for
o Assay development support for...
APC instruments are highly flexible
11
Recordings from primary cells possible
300 nM Haloperidol
Negative control
Neurons ...
Modes of operation
12
Excised patch recordings not (yet, really) possible by planar APC
"Whole-
cell"
• Most widely used f...
o Different features available
o Voltage clamp, current clamp (action potential recordings)
o Whole cell, perforated patch...
Thank you for your attention and input!
Andreas Ebneth
Rainer Netzer
Heike Deisemann
Desireé Amm
York Rudhard
John Kemp
Th...
References & Recommended Reading
15
References:
- Clemens Möller (2010). Keeping the Rhythm: hERG and beyond in Cardiovasc...
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Nanion Usergroup Meeting Sept 2011

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Automated Patch-Clamping. Presentation at the Nanion Usergroup Meeting, Munich, Sept 2009

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Nanion Usergroup Meeting Sept 2011

  1. 1. Round Table Discussion Thoughts on APC assay optimization - Strengths and weaknesses of APC instruments Prof. Clemens Möller, PhD Albstadt-Sigmaringen University of Applied Sciences office@biophysicalconsulting.de www.clemensmoller.de Nanion Usergroup Meeting Sept 29, 2011
  2. 2. Correlation MPC-APC Assay set up Flexibility of operation; primary cells 2 Round Table Discussion
  3. 3. Controlled state of channel Low binding of hydrophobic compounds Low leak currents Control of membrane potential / capacitance Patch- Clampers desire to tinker with the experiment PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC Continuous voltage control Chips made of glass substrate Gigaseals RS compensation HEKA Software for experiment and data evaluation 3 Experiments are performed under similar conditions as in MPC
  4. 4. Controlled state of channel Low binding of hydrophobic compounds Low leak currents Control of membrane potential / capacitance Patch- Clampers desire to tinker with the experiment PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC Continuous voltage control Chips made of glass substrate Gigaseals RS compensation HEKA Software for experiment and data evaluation 4 Experiments are performed under similar conditions as in MPC Main difference to MPC: Cells are delivered from suspension (not adherent).  Pharmacology? Networks of cells?
  5. 5. Potential (mV) -100 -50 0 -2 -1 0 1 Peak current (nA) . potential (mV) -140 -120 -100 -80 -60 -40 -20 0 20 40 current(nA) -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 . Biophysical characterization of hERG channels 5 Manual Patch-Clamp PatchLiner Current-voltage relationships of hERG channels correlate very well Reference: Möller and Witchel (submitted).
  6. 6. 6 Excellent correlation between manual and planar patch clamp Before employing automated (planar) patch-clamping in our programs, the devices were validated with reference and actual program compounds. 57 compounds within one chemical series were tested side by side on manual rigs and a planar patch clamp device (PatchLiner) Only 5 out of 57 compounds showed a difference in the IC50-values of ~5-fold Correlation: Manual Patch-Clamp vs PatchLiner Manual Patch-Clamp IC50 [µM] 0.1 1 10 100 PatchLinerIC50[µM] 0.1 1 10 100 5 52 Pharmacological comparison of Electrophysiology Platforms PatchLiner validation Reference: Davenport et al., 2010
  7. 7. Key points for pharmacology: same as for MPC and HT systems o Prepare compound solutions as freshly as possible. Observe solubility of compounds. o Compound stock solutions required? How long are the compounds stable in DMSO, at which storage temperature? o Store solutions with reduced vehicle (DMSO) content in glassware, for as short period as possible. o Are currents stable under negative control conditions? Any vehicle effects? o Do currents reach steady state in presence of compounds? (No continuous perfusion in APC; repeated cpd administration required?) o Consider pulse protocols (do the compounds exhibit preference for certain states of the channel?) o Consider temperature effects 7 PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC
  8. 8. (Many) Port-A-Patch, PatchLiner and SyncroPatch assays are easy to set up o Operation in standard modes easy o Very "forgiving" cell culture o Good seal success rates can be achieved with suboptimal cells o But: Seal enhancer (for most cell types) appears to be required for good success rates? 8 Eccellent seal success rates Kv1.5 HERG 1st tier profilingExample: Panel of cardiac channels on PL Kv1.1 Kv4.3/KChIP2 NaV1.5 L-type Ca2+ Standard protocols for most cell types and channels are available. For many cells, excellent success rates can be achieved. Current traces from Möller et al., 2010
  9. 9. (Many) Port-A-Patch, PatchLiner and SyncroPatch assays are easy to set up o The healthier the cells are, the better the seal success rate will typically be o Cell confluency ~60-80%. Can depend on cell type o Especially small / large cells?  Consider different chip hole size o Cell density appears to be not so critical (1 x 106 – 5 x 107 cells/ml are good standard densities, but much lower densities have worked fine for some cells) o Relatively small effect of pressure etc. settings in PatchControl software; standard settings are often a good choice 9 Key points to consider for a good seal success rate
  10. 10. APC instruments complement each other In addition, the Port-A-Patch proved very useful for o Assay development support for PatchLiner and SyncroPatch o Verification of data for compounds that showed inconsistent IC50 values on the PatchLiner or the SyncroPatch 10 Instruments for different needs of throughput Port-a-Patch PatchLiner SyncroPatch
  11. 11. APC instruments are highly flexible 11 Recordings from primary cells possible 300 nM Haloperidol Negative control Neurons – MAP2 astrocytes – GFAP Nuclei – DRAQ5 Neurons Cardiomyocytes Na+ Ca2+ K+ Reference: Möller et al., 2010
  12. 12. Modes of operation 12 Excised patch recordings not (yet, really) possible by planar APC "Whole- cell" • Most widely used for pharmacology "Cell- attached " • Possible with some cells "excised patch" • Are you missing single channel recordings by APC? For MoA?
  13. 13. o Different features available o Voltage clamp, current clamp (action potential recordings) o Whole cell, perforated patch (are you using this a lot?) o Intracellular solution exchange o Fast ligand exchange (~50 ms) o Temperature control o Interaction during experiment possible o Patch-Clampers desire to "play around" with an experiment o Also, a "screening mode" with limited user access to settings is possible (Talk by Corinna from last years meeting) Different features available APC instruments are highly flexible 13 Many features available; interaction possible Interaction during experiment possible
  14. 14. Thank you for your attention and input! Andreas Ebneth Rainer Netzer Heike Deisemann Desireé Amm York Rudhard John Kemp The whole great team, especially: Niels, Andrea, Michael, Claudia, Sonja, Timo, Ali & Cecilia Ralf Kettenhofen Martin Stolz Prof. Clemens Möller, PhD | Albstadt-Sigmaringen University of Applied Sciences clemens@biophysicalconsulting.de | www.clemensmoller.de 14 Carsten Claussen Clemens Möller | Albstadt-Sigmaringen University of Applied Sciences office@biophysicalconsulting.de | www.clemensmoller.de
  15. 15. References & Recommended Reading 15 References: - Clemens Möller (2010). Keeping the Rhythm: hERG and beyond in Cardiovascular Safety Pharmacology. Expert Reviews Clinical Pharmacology (Ion Channels) 3: 3. 321-329 May - Adam J Davenport, Clemens Möller, Alexander Heifetz, Michael P Mazanetz, Richard J Law, Andreas Ebneth, Mark J Gemkow (2010). Using Electrophysiology and in silico 3D Modelling to reduce hERG inhibition in a Histamine H3 Receptor Antagonist Program. ASSAY and Drug Development Technologies 8: 6. 781-789 December - Clemens Möller, Mark Slack (2010). Impact of new technologies for cellular screening along the drug value chain. Drug Discovery Today 15: 9-10. 384-390 May - Clemens Möller, Harry Witchel. Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening. (Submitted to Frontiers, 2011) Recommended reading: Carol J Milligan, Li J, Sukumar P, Majeed Y, Dallas ML, English A, Emery P, Porter KE, Smith AM, McFadzean I, Beccano-Kelly D, Bahnasi Y, Cheong A, Naylor J, Zeng F, Liu X, Gamper N, Jiang LH, Pearson HA, Peers C, Robertson B, Beech DJ (2009). Robotic multiwell planar patch-clamp for native and primary mammalian cells. Nat Protoc. 2009;4(2):244-55.

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