Ion Channels

Quality Cells for High Performance Channel Assays

 

Conduct quality, highly reproducible ion channel assays by rapidly expressing functional ion channels – even toxic or intractable channels — in cells with a high level of membrane integrity. Assay results are comparable to stable cell lines, yet assays can be developed and conducted in just a fraction of the time. Cryopreserve cells to create an assay ready cell bank without sacrificing performance.

  • Cotransfect multiple plasmids for expression of multi-subunit channels
  • Eliminates the need for stable cell lines
  • >90% transfection efficiency and >90% cell viability for most cell types
  • Use physiologically relevant cell types including primary or stem cells
  • Fully scalable, able to transfect 5×105 cells in seconds up to 1×1010 cells in < 30 minutes to support assay development through screening campaigns

Applications

Manual and automated patch clamp assays including Molecular Devices, Nanion & Sophion platforms

Flux assays such as thallium or FLIPR®

Sodium channels

Potassium channels

Calcium channels

Transporters

 

 

Multi-subunit Calcium Channel Expression

 

HEK293 cells were cotransfected with four cDNAs encoding the pore-forming alpha subunit of the voltage-gated calcium channel Cav2.2, the modulatory β subunit, the modulatory α2δ subunit, and an inward rectifier potassium channel (Kir2.1). Calcium influx FLIPR assays were conducted 24 hrs post transfection on untransfected HEK 293 cells, cells transfected with 3 calcium channel subunits ± the inward rectifier. Dye was added in a low potassium, low calcium solution and loaded for 30 minutes. Vehicle control or a calcium channel specific antagonist (600 nM ω-Conotoxin GVIA) was added for an additional 30 minutes. Cells were depolarized with high external K+ (up to 135 mM). Data courtesy of Charles River Laboratories (formerly ChanTest Corporation).

High Throughput Potassium & Sodium Channel Variant Screening

 

Flow cytometry results for channel expression and cell viability for (A) KCNE1 cell line transfected with two different concentrations of KCNQ1_IRES2_EGFP, and (B) CHO-K1 cells co-transfected with KCNQ1_IRES2_EGFP and KCNE1_IRES2_DsRedMST constructs. Automated patch clamp assays were run on the Nanion SyncroPatch 768PE results. Example results (C) for cells co-transfected with KCNE1 and various KCNQ1 variants, (D) modified ND7/23 cells transfected with SCN1A or SCN5A. Data Courtesy of Dr. George Laboratory, Northwestern Medicine.

Quality Assay Ready Cells Through Cryopreservation

A. Nav1.5 Activity Before Cryopreservation

* Measured @ 0 mV in simple depolarizing step protocol
** Percentage block compared to saline period of a 20 mM TTX single addition

B. Nav1.5 Activity Post Cryopreservation

* Measured @ 0 mV in simple depolarizing step protocol
** Percentage block compared to saline period of a 20 mM TTX single addition
HEK 293 cells were transfected with 1.5 µg/1e6 cells of a Nav1.5 expression plasmid. Transfected cells were cultured for 24 hours at 37°C. Half of the cells were transferred to 28°C and half were kept at 37°C for an additional 24 hours. Half the cells were assayed on the Sophion Qpatch in single hole mode 48 hours post transfection; remaining cells were cryopreserved and assayed immediately after thawing.

Comparable Results Using Transiently and Stably Expressed Potassium Channel

CHO K1 cells transiently transfected with a Kv1.5 expression plasmid or a CHO stable cell line expressing Kv1.5 were incubated with varying concentrations of three potassium channel inhibitors and assayed on the PatchXpress from Molecular Devices.  A). Pharmacological analysis of transiently transfected cells using the PatchXpress instrument.  B). Representative current tracings that depict a step-wise loss of Kv1.5 activity in response to increasing concentrations of the inhibitor capsaicin. Table: IC50 values for each compound compared favorably to data obtained using a stable cell line. Data courtesy of BioFocus.

CFTR Corrector & Modulator Screening in Biologically Relevant Cells

 

CFTR-YFP Iodide Flux Assay with Transiently Transfected Human and Rat Epithelial Cells A). Images of either CFBE (human bronchial epithelial) cells co-transfected with a YFP expression plasmid and CFTR-dF508 plasmid or FRT (fisher rat thyroid) cells stably expressing YFP and CFTR-dF508. B). High throughput iodide flux assay responses of cells co-transfected with YFP and CFTR-dF508 plasmids or FRT cells stably expressing CFTR-dF508 to various concentrations of C-18, a known CFTR corrector. dQR = change is the YFP quenching rate. Data courtesy of Flatley Discovery Lab.

High Throughput, Human TRPV1 Screening

High Z’ Factors for TRPV1 Screening of Transiently Transfected, Cryopreserved Cells. Cells were transiently transfected with an expression plasmid (2μg/1E6 cells) encoding human TRPV1 and the cells were cryopreserved.  Transfected cells were assayed immediately post thaw.  Cells were incubated with a known agonist and antagonist and responses measured.  Z’ factors were determined.

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