RECOMMENDED CULTURE CONDITIONS Recommended culture conditions and standard operating procedure are provided with the product.

Transcription

1 PRODUCT DATASHEET PrecisION hnav1.5-hek Recombinant Cell Line Catalog Number: CYL3004 PRODUCT DESCRIPTION Recombinant HEK293 cell line expressing the human Nav1.5 (type V voltage-gated sodium channel alpha subunit, accession number NM_000335). ASSOCIATED PRODUCTS The PrecisION hnav1.5-hek293 Recombinant Cell Line is provided to customers on the purchase of an appropriate license. The available licenses are: CYL3004SS PrecisION hnav1.5-hek Single Site License CYL3004TS PrecisION hnav1.5-hek Two Site License CYL3004MS PrecisION hnav1.5-hek Multiple Site License CONTENTS 2 x 1 ml aliquots containing 1.32 x 10 6 cells/ml in 10% DMSO at passage 10. STORAGE Vials are to be stored in liquid N 2. WARNINGS For Research Use Only; Not for Use in Diagnostic Procedures Not for Animal or Human Consumption GMO This product contains genetically modified organisms. Este producto contiene organismos genéticamente modificados. Questo prodotto contiene degli organismi geneticamente modificati. Dieses Produkt enthält genetisch modifizierte Organismen. Ce produit contient organismes génétiquement des modifiés. Dit product bevat genetisch gewijzigde organismen. Tämä tuote sisältää geneettisesti muutettuja organismeja. Denna produkt innehåller genetiskt ändrade organismer. MYCOPLASMA TESTING The cell line has been screened using the ELISA based Mycoplasma Detection kit (Roche) and by a PCR VenorGem kit (Minerva Biolabs) to confirm the absence of Mycoplasma species. FUNCTIONAL VALIDATION The PrecisION hnav1.5-hek recombinant cell line was analyzed by patch-clamp techniques giving typical peak currents between na. With IonWorks, typically greater than 80% of cells expressed peak currents of an average amplitude of na. RECOMMENDED CULTURE CONDITIONS Recommended culture conditions and standard operating procedure are provided with the product. Eurofins Pharma Bioanalytics 15 Research Park Drive T Services US Inc. St Charles MO F USA Template version: 6. Modified Date: 21JUL14. Modified by: SGJ

2 FUNCTIONAL VALIDATION Electrophysiological properties of the hnav1.5 current. Whole-cell Patch Clamp Electrophysiology. Figure 1. Representative currents produced by the PrecisION hnav1.5-hek cell line. A. Representative current traces elicited by a series of test pulses given in 10 mv increments from the holding potential of -90 mv. Typically peak currents range between na (no currents are recorded in untransfected cells). B. The current-voltage relationship shows currents begin to activate at approximately 50 mv, reach a peak at 20 mv and have a reversal potential (E rev ) of ± 4.6, n=3 (calculated E rev +37 mv). 2

3 Figure 2. Whole cell patch clamp recordings of the PrecisION hnav1.5-hek cell line. A. Shows a series of currents evoked by 20 ms depolarizing steps from a holding potential of 110 mv. B. The peak current-voltage relationship. The observed reversal potential of +60 mv is close to the theoretical Na + equilibrium potential of +58 mv under the ionic conditions. C. Shows the steady state inactivation curve for hnav1.5, constructed from 4 s conditioning pulses to the voltages shown on the abscissa, followed by test pulses to 20 mv. The V ½ for inactivation was 90.1 mv and the slope value (k) was A B mv pA -600 C ms -800 pa V h -110mV I/I Max 0.4 V mV Slope V h -120mV 4s conditioning Conditioning potential 3

4 Figure 3. Use of the PrecisION hnav1.5-hek cell line in FLIPR screening assays. In hnav1.5-hek cells incubated with DiBAC and scorpion venom (ScTx), KCl induces reproducible membrane potential depolarizations (increased fluorescence) that are dependent in size upon the concentration of ScTx used. No responses are seen in untransfected HEK293 cells. Figure 4. Use of the PrecisION hnav1.5-hek cell line in VIPR screening assays. In hnav1.5-hek expressing cells incubated with VSP fluorescent dyes (voltage sensitive probes) and scorpion venom, a rapid Na + ion-dependent membrane potential depolarizations (increase in the relative ratio of fluorescence from the 2 VSP dyes) are observed. These are significantly greater than in untransfected controls and can be blocked with the standard inhibitor tetracaine. 4

5 Figure 5. Use of the PrecisION hnav1.5-hek cell line in IonWorks HT screening assays. A. Simultaneous recording of currents from 48 wells of an IonWorks HT patch plate, each trace represents currents recorded from an individual hnav1.5-hek cell. B. Typically greater than 80% of cells express peak currents with an average amplitude of 2.4 na. C. Concentration response curves for 2 standard inhibitors tested by IonWorks HT. Stability of PrecisION hnav1.5-hek Cell Line: The PrecisION hnav1.5-hek cell line has stable expression for >25 passages. 5

6 Vector: HpaI ClaI NdeI BssHII AscI XhoI NotI NsiI SspI NdeI MluI NaeI BamHI KpnI IVS CMV promoter EcoRI BclI Sse8387 XhoI MluI AgeI Amp BsaBI EcoRI 8000 polya XhoI Neo BclI XbaI NaeI BssHII TthI NarI KpnI IRES SfiI TthI pcin5-hnav bps hnav1.5 KpnI PshAI BamHI NaeI NarI NdeI NheI SmaI XmaI BamHI Polylinker: CMV-IVS-NotI-Nav1.5-NotI-EcoRI-IRES-neo hnav1.5 Sequence: The sequence of the cdna used to make this cell line contains a coding two base pair mutation with respect to the accession number NM_ GGC to GCT - at positions conferring the amino acid change Gly180Ala. 6

7 REFERENCES 1. Clare, J.J., Tate, SN., Nobbs M. and Romanos, M.A. (2000) Voltage-gated sodium channels as therapeutic targets. Drug Disc. Today 5: Gellens, M.E., George, Jr. A.L., Chen, L., Chahine, M., Horn, R., Barchi, R.L. and Kallen, R.G. (1992) Primary Structure and Functional Expression of the Human Cardiac Tetrodotoxin-Insensitive Voltage-Dependent Sodium Channel. Proc. Natl. Acad. Sci. USA 89 (2): Schroeder, K., Neagle, B., Trezise D.J. and Worley, J. (2003) Ionworks HT: a new high-throughput electrophysiology measurement platform. J. Biomol Screen 8 (1): FOR RESEARCH USE ONLY; NOT FOR USE IN DIAGNOSTIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. No part of these works may be reproduced in any form without permission in writing. Eurofins Pharma Bioanalytics Services US Inc. is an independent member of Eurofins Discovery Services 7