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Efficient transfection of mammalian primary and stem cells using the Neon™ Transfection System
Sponsored,vendor-submitted protocol    Sponsored by Invitrogen Corporation, part of Life Technologies    Published in November 2009 (p.29) DOI: 10.2144/000113288

Abstract

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.

Materials

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)

Methods

Table 1. Optimized protocols for stem cells and primary Cells with the Neon™ Transfection System

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.

Optimization protocol

Figure 1. Fluorescence images of transfected cells. Adipose-derived stem cells (A), rat fetal neural stem cells (B), primary human macrovascular endothelial cells, neonatal dermis (C), and primary human dermal fibroblasts, neonatal (D) were transfected using the Neon™ Transfection System and 0.5 μg of a plasmid encoding the EGFP. Cells were analyzed by fluorescence microscopy 24–48 h after transfection.

Please see the 24-well Optimization protocol within the full-length version of this protocol available online at www.biotechniques.com/protocols/113288.

Analysis

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.

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