Volume 4, September 2010

IN THIS ISSUE

Featured Application
Functional Ion Channel Screening

Excerpts from an STXpert
Ion Channel Q&A Session with Dr. Barbara Wible, Head of Cell and Molecular Biology at ChanTest.

Upcoming Meeting
Join us at Miptec
September 20-24, 2010
Booth #G16
Workshop Sept 13, 12:45 PM

Frequently Asked Questions
How do you transfect multiple plasmids at the same time with the MaxCyte STX?

MaxCyte’s proprietary Flow Electroporation technology leads the way in high performance, high throughput transfection enabling the widest array of cost and time effective drug discovery assays. MaxCyte technology lets you get ahead by transfecting any molecule of interest, into any cell, at any scale. The goal of the MaxCyte Minute is to provide researchers like you with key application and scientific advancements to help you stay ahead in the world of drug discovery.

Featured Application – Functional Ion Channel Screening

Multi-subunit Ion Channel Expression via Transient Transfection

Increased attention is being paid to voltage and ligand-gated ion channels as higher throughput, cell-based assays of channel activity have emerged. Current ion channel assays, such as automated patch clamp and FLIPR^® assays, rely heavily on the use of stable cell lines, which are challenging to create due to their multi-subunit nature as well as requiring months to develop. Furthermore, ion channels can be toxic when expressed at high levels, creating the need for inducible promoters, which adds an additional layer of complexity. Transient transfection offers an attractive alternative.

Using the MaxCyte STX™ Transfection System, multiple expression plasmids can be co-transfected in defined stoichiometric ratios to produce specific functional ion channel complexes. Both ligand-gated and voltage-gated ion channels have been successfully expressed in a variety of cell types and shown to be functional via calcium flux and electrophysiology assays.

ChanTest^®, a leader in developing and conducting Ion Channel Assays, routinely uses the MaxCyte STX within their assay protocols. For an example of their data from a functional cell-based assays using the Cav2.2 calcium channels…click here

Excerpts from a MaxCyte STXpert

Ion Channel Q&A Session with Dr. Barbara Wible, Head of Cell and Molecular Biology at ChanTest.

Dr. Wible is an industry expert in ion channels and currently holds the position of Head of Cell and Molecular Biology at ChanTest, a leading Ion Channel and GPCR contract service organization. During a webinar presented by MaxCyte and ChanTest, Dr. Wible addressed a number of questions from participants. We have chosen an excerpt of these questions and have formatted them in a Q&A format. These questions and more are discussed during the second half of a webinar entitled: Improved Transfection of Ion Channels and Other Targets for Cell Based Assays.

Q: During your initial evaluation of the MaxCyte STX, what ion channels and assays did you run?

A: We wanted to put the MaxCyte STX to the test so we chose to focus on development of a FLIPR assay for voltage-gated calcium channels. This required transiently coexpressing four different ion channel cDNAs within cells in order to generate a robust calcium influx FLIPR assay.

Q: Are ion channels inherently difficult to work with?

A: Getting a good FLIPR assay for voltage-gated calcium channels can be very difficult in HEK and CHO cells especially for Cav2.1, Cav2.2, and Cav3.2. The channels are largely inactivated at the normal resting potential of the HEK cells (-40 or -50 mV). Adding an inward rectifier potassium channel is the only way to get a robust assay.

Q: Why did you decide to look at these ion channels first? Is there a specific advantage to transiently transfecting these subunits over constitutive or inducible stable expression?

A: We wanted to see whether the MaxCyte STX would provide a viable alternative to construction of a stable cell line for a voltage-gated calcium channel assay in FLIPR. This involved cotransfecting 4 cDNAs (one of which was a 5-6 kb insert). It also allowed us to see how the MaxCyte STX would handle the transfection of multiple subunits.

We express Cav channels in inducible systems as constitutive expression of Cavs is toxic due to calcium entering the cells. When expressed alone, we also express the inward rectifier in an inducible system as constitutive expression can slow cell growth. Working with an inducible system adds an extra step (induction) prior to assay that is avoided when all subunits are expressed transiently.

Q: How long did it take you to get a working assay using the MaxCyte STX?

A: Very quickly with HEK293. CHO cells are more difficult to transfect in general, require more DNA (with consequently lower cell viability), and often result in less functional expression of ion channels than HEK293.

Q: Is the instrument easy to use? How much DNA is needed for each transfection and does the DNA need to be of special quality?

A: The instrument is very easy to use. Programs for commonly used cells are already available so for HEK293 cells all that was necessary was to mix the DNA and cells in the transfection unit and run the instrument. Initially, we did a titration with GFP to assess the transfection efficiency with our particular batch of HEK cells and then used the amount of DNA that gave the best transfection efficiency with the least cell death.

Q: Why would you use the MaxCyte system versus other transfection systems?

A: For ChanTest, the ability to transfect large batches of cells is key. For FLIPR screening, large numbers of cells are needed. Also, for automated patch clamp including PatchXpress®, QPatch HT, and especially IonWorks® Quattro, between 1-6 million cells need to be loaded onto the instrument even though a much smaller number 16, 48, or 384 recordings are made per run per instrument.

Related Ion Channel Resources

	Webinar presented in conjunction with ChanTest: Improve Transfection of Ion Channels and Other Targets for Cell Based Assays. View the webinar
	Webinar in conjunction with BioFocus: Improve Transfection of Ion Channels for Cell-Based Assays. View the webinar
	Application Note 3: Rapid Production of Cells for Screening Voltage-Gated Ion Channels in Automated Electrophysiology Assays Using the MaxCyte STX Scalable Transfection System. Download
	Poster, SBS 2010, Phoenix, AZ: Creating Cell Based Assays for Screening GPCRs, Ion Channels and Other Targets in Cell Lines and Primary Cells Using the MaxCyte STX Scalable Transient Transfection System. Download
	Poster, MipTec 2009, Basel, Switzerland: Rapid Automated Development of Cell Based Assays for Screening GPCRs, Ion Channels and Other Target Molecules Using the MaxCyte STX Scalable Transient Transfection System. Download

Come See MaxCyte at Miptec 2010

September 20-24, 2010, Basel, Switzerland - Booth #G16

MaxCyte Symposium
Thursday, September 23, 2010
12:45 – 1:45pm, Lecture Hall F

Optimizing Cell Based Assays from the Bench to HTS Using Scalable Transient Transfection
Dr. James Brady, Director of Technical Applications, MaxCyte



MaxCyte is also a proud sponsor of:
The Eukaryotic Protein Expression Workshop
Monday, September 20, 2010
A part of the BIOVALLEY Life Sciences Week

Frequently Asked Question

How do you transfect multiple plasmids at the same time with the MaxCyte STX?

The MaxCyte STX can be used for the simultaneous transfection of multiple expression plasmids as well as multiple agents such as DNA, RNA, siRNA, proteins, and small molecules. Transfection of multiple plasmids or agents is accomplished by simply mixing the various agents at the desired ratios in a single solution using MaxCyte’s universal transfection buffer. We recommend beginning with an equimolar ratio of plasmids and conducting subsequent optimization of plasmid ratios based upon the assay response.

For more information about these applications or the MaxCyte STX Scalable Transfection System
Contact Us at: info@maxcyte.com • +1 301 944 1700
Or Visit Us at: www.maxcyte.com

ChanTest is a registered trademark of ChanTest. MaxCyte is a registered trademark of MaxCyte. MaxCyte STX is a trademark of MaxCyte. FLIPR, PatchXpress, and IonWorks are registered trademarks of Molecular Devices, Inc. iPod Shuffle is a registered trademark of Apple, Inc.