The Neon™ Transfection System is an improved transfection technology designed for highly efficient gene delivery into many cell types that are difficult to transfect, including primary and stem cells. The Neon™ technology uses gold-plated electrodes in a narrow, plastic pipette tip for the electroporation chamber, resulting in a more uniform electric field with minimal pH changes. The unique chamber design simplifies the workflow and provides high-transfection efficiencies and cell viability compared with methods using a traditional cuvette-based electrode.
Human microvascular endothelial cells, neonatal dermis (C010-5C; Invitrogen, Carlsbad, CA, USA)
Human dermal fibroblasts, neonatal (C004-5C; Invitrogen)
StemPro® human adipose-derived stem cells (R7788-115; Invitrogen)
GIBCO® rat fetal neural stem cells (N7744-100; Invitrogen)
Dulbecco’s phosphate-buffered saline (DPBS) (1×), liquid, no calcium or magnesium (14190; Invitrogen)
TrypLE™ Express (12563; Invitrogen)
StemPro® Accutase® (A11105; Invitrogen)
Vivid Colors™ pcDNA™6.2/EmGFP plasmid DNA (K35920 or K36020; Invitrogen)
Neon™ Transfection System 10 µL Kit (MPK-1096; Invitrogen)
24-well tissue culture–treated plates (Cat. no. 3524; Corning, Lowell, MA, USA)
Prepare 24-well plates by adding 500 µL culture medium containing serum (without antibiotic) and pre-incubate plates in a humidified 37°C/5% CO2 incubator.
1. Aspirate the medium from the cells and rinse the surface of the cell layer with DPBS, without calcium or magnesium.
2. Aspirate the DPBS and discard.
3. Detach the human macrovascular endothelial cells (HMVECnd), HDFn, and adipose-derived stem cells (ADSCs) with TrypLE™ Express and detach the rat neural stem cells (rNSCs) with StemPro® Accutase.
4. Transfer the dissociated cells to a 50-mL conical tube and centrifuge at 100–300× g for 5 min at room temperature.
5. Aspirate and discard the medium.
6. Carefully resuspend the cells in 10 mL DPBS, without calcium or magnesium.
7. Determine the total number of cells and percent viability.
8. Transfer 3.0 × 106 cells to a new tube and centrifuge at 100–300× g for 5 min at room temperature.
9. Aspirate and discard the DPBS.
10. Carefully resuspend the cells in 285 µL Resuspension Buffer R to achieve the desired cell concentration. Recount the cells.
11. Add plasmid DNA (15 µg) to resuspended cells.
12. Fill one Neon™ tube with 3 mL Solution E.
13. Load 10 µL of the cell and DNA mixture into an electroporation tip using the Neon™ pipet and insert into the pipet station.
14. Load the 24-Well Optimization protocol or alternatively set the instrument parameters for voltage, width, and pulse, using the appropriate Neon™ protocol.
15. Press the Start button.
16. After the pulse, remove the Neon™ pipet from the pipet station.
17. Transfer the sample into the prepared 24-well plate.
18. Repeat steps 14–18 for the remaining samples.
Please see the 24-well Optimization protocol within the full-length version of this protocol available online at www.biotechniques.com/protocols/113288.
Cells were analyzed for viability and GFP fluorescence after 24 or 48 h using a Guava PCA-96 Cell Analysis System. Live/dead cell populations were determined using PI staining. Transfection efficiency was calculated based on the total population.
Results and discussion
Using the 24-well Optimization protocol, we identified parameters that produced high-transfection efficiency and cell viability for several mammalian stem cell types, including rNSCs and human ADSCs. High-transfection efficiencies were also obtained with several primary cell types, including HMVECnd and human neonatal dermal fibroblasts (HDFn). Additional improvement in transfection of the two primary types was observed after a second round of optimization to further refine parameters.
Any proper birthday celebration must begin with thanks. BioTechniques’ roster of gratitude starts with our readers, authors, and reviewers (who often switch from role to role), whose services to—and very evident affection for—the journal keep us going. We thank our advertisers, who have made it possible for us to fulfill our mission for the past 25 years (and, we hope, for at least 25 more).
For this, our Anniversary Issue, we express special thanks to those on our Editorial Board, who, in addition to their year-in and year-out counsel, have provided us this month with a score of reviews of some of the most powerful tools in biological research.Elegance
A thing is elegant when it takes its most compact form, when every piece is vital and nothing is superfluous.
The Brooklyn and Golden Gate bridges are elegant. An evening gown or a Grand Banks schooner under full sail may be elegant. The identity eiπ = −1 is elegant.
Business ideas may be elegant, too—though the chaos of getting and spending can obscure the purity of line.
Here is the world that faced the publishers of BioTechniques in 1983: new tools of molecular biology had unleashed a flood of new data. These fueled hundreds of new insights, and biology seemed to shatter into scores of tight subdisciplines. In each, researchers dug into ever-deeper detail. Every disciplinary schism bred a newer, narrower, denser journal. It sometimes seemed that the journals’ very focus threatened to cut these pockets of research off from one another.
And yet, the tools scientists used were not “disciplined” and could not be confined to silos. Immunoaffinity. SDS-PAGE. Cell culture. Labeling. DNA sequencing. Peptide synthesis. Gene amplification. The methods transcended disciplines.A Journal for Everyone
Biologists needed a common ground on which they could trade tools—a peer-reviewed journal that would be useful to all.
But how can you reach everyone and still keep publishing? The general business model was then (and still remains, despite pressure for change) the high-subscription-price, low-circulation journal. But that was the antithesis of what the founders of BioTechniques intended, and it was not the way to bind together the disciplines of molecular, developmental, and cellular biology.
So, true to the spirit of the field it wanted to serve, BioTechniques created itself as a hybrid—with the head and heart of a peer-reviewed journal and the sinews of a controlled-circulation, advertising-supported magazine. From the beginning, BioTechniques has been distributed free to researchers.
I first came to know BioTechniques as a competitor, soon after it was founded. Even then I acknowledged—publicly if grudgingly—the elegance of the conception. BioTechniques was open access when open access wasn't cool—before, in fact, “Open Access” was even created—with a sustainable model that does not charge individual readers for reading *There is a charge for institutional subscriptions.
*There is a charge for institutional subscriptions.
Over the past year, we have consolidated our Open Access credentials: we eliminated the last of our author charges (for color plates) and amended our copyright agreement so that we take a license from our authors, but leave the copyright with them.
We have added BioTechniques Weekly, a newsletter to bring readers breaking news on research issues and funding opportunities. We have added a digital edition of our journal to make BioTechniques available in regions of the world where distributing paper copies would be prohibitively expensive.
We are rebuilding BioTechniques.com to provide more tools and information than ever before, while making the information easier to find and easier to use, from our archive of peer-reviewed papers to protocols to interactive forums on technique and method.
All of this serves the original vision: to make the tools of molecular biology available to everyone. And for that vision, on our birthday, we close with one last note of thanks, to the founders of BioTechniques and to all of those who have served the journal from 1983 until today.
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