Optimizing Transfection Protocols Using the Neon® Transfection System and the Tali® Image Cytometer
Scientists’ ability to manipulate cells via transfection with nucleic acids, proteins, and siRNA has led to major scientific advancements. However, introducing these molecules into difficult-to-transfect cells, and the lack of tools available to easily and accurately quantify transfection efficiencies, have frustrated scientists for years.
The Neon® Transfection System and the Tali® Image Cytometer are two benchtop platforms that can help solve these critical issues. The Neon® Transfection System provides fast and efficient delivery of nucleic acids into all mammalian cell types, including primary, stem, and difficult-to-transfect cells (DTTCs). The Tali® Image Cytometer delivers quick, accurate, and quantitative measurements of transfection efficiencies of GFP and RFP vectors.
GFP/RFP Transfection & Cell Viability on Tali
In this application note, we demonstrate how the Neon® Transfection System and the Tali® Image Cytometer can be used in tandem to help quickly optimize your transfection. The Neon® Transfection System is preprogrammed with a 24-well optimization protocol to help users identify the optimal transfection efficiency for each cell type. The Tali® cytometer can quantitatively measure GFP expression and cell viability of samples transfected using each of the 24 Neon® system settings. Data from the two devices facilitate selection of transfection parameters that deliver the optimal combination of GFP expression and cell viability required for downstream applications.
Materials and Methods
Jurkat cells were transfected using a pcDNA™ 6.2 emGFP plasmid containing an EF-1α promoter, at a concentration of 1 µg/µL. Briefly, Jurkat cells were cultured in RPMI medium supplemented with 10% FBS and 2 mM glutamine, at 37°C and 5% CO2. The cells were harvested, spun down (7 min at 2,000 rpm), resuspended in PBS (without Ca2+ or Mg2+), counted, and then resuspended in Neon® resuspension buffer R at a concentration of 2 x 107 cells/mL. The cell solution (300 µL) was combined with 30 µL of EF-1α emGFP plasmid (1 µg/µL). The volume of the mixture was more than sufficient to transfect cells using the 24 different conditions in the Neon® electroporator optimization protocol using the 10 µL tips with 2 x 105 cells and 1 µg of DNA per well. Cells were then electroporated with the Neon® Transfection System using the recommended 24 combinations of pulse voltage, pulse width, and pulse number. Following electroporation, each sample was dispensed into a well of a 24-well microplate containing 300 µL of prewarmed growth medium and incubated for 24 hr in routine culture conditions. The next day, cultures were treated with 50 µL TrypLE™ Select cell dissociating reagent to remove cell clumps, and 150 µL of dissociated cells were combined with Tali® Dead Cell Red stain (included with the Tali® Viability Kit) and lightly pipetted up and down to mix thoroughly. The cells were then analyzed on the Tali® cytometer.
The Tali® cytometer is capable of measuring cellular fluorescence that falls within one of its fluorescence channels: green (458 nm excitation with a 525/20 nm emission filter) or red (530 nm excitation with a 585 nm longpass emission filter). The green channel of the Tali® cytometer is optimized for GFP detection. The fluorescent Dead Cell Red stain in the Tali® Viability Kit is matched to the red channel of the instrument. The viability assay uses a minimal amount of cell sample and can typically be performed in culture medium in less than 5 minutes.
The Neon® Transfection System and the Tali® Image Cytometer were used to optimize the electroporation settings for transfecting Jurkat cells with a GFP-encoding plasmid. Using the preprogrammed 24-well optimization protocol of the Neon® Transfection System (Figure 1A), a range of transfection efficiencies were seen (Figure 1B), demonstrating the importance of optimized electroporation settings for maximal delivery of plasmid into cells. The optimization function systematically evaluates a range of voltages, pulse widths, and number of pulses. After 24 hours we found GFP-expressing cells in all but the untransfected control well, both visually by microscopy and with the Tali® cytometer.
|Figure 1. Optimization protocol on the Neon® Transfection System. (A) The Neon® Transfection System screen showing the built-in optimization parameters that can be readily selected with a single touch. (B) Table from the Neon® Transfection System manual showing the 24 settings used for optimization. This table was used to determine the settings for the Neon® Transfection System that produced optimal transfection efficiencies and cell viability as determined by the Tali® Image Cytometer.|
The Tali® Image Cytometer was used to quantitate the percentage of cells expressing GFP. The cell-size gate on the cytometer was used to exclude debris for the fluorescent analysis. GFP-expressing cells were distinguished from non-expressing cells by setting a minimum fluorescence value (thresholding) on the histograms generated by the Tali® cytometer. The threshold was set to the right edge of the untransfected control histogram. The fluorescence thresholds were then visually confirmed using the bright-field cell image overlays in each fluorescent channel, which indicated how each individual cell was categorized by fluorescence (Figure 2).
The Dead Cell Red stain (propidium iodide) was used to obtain a quantitative measurement of cell viability in parallel with GFP-expression measurements. Cells that excluded the stain were considered viable. Similar fluorescence thresholding on the red histogram is all that is required to get this measurement. Jurkat cells remained viable after all but the most extreme Neon® ystem transfection voltage settings. The percentage of viable cells ranged from 72% to 92%, with 21 of 24 transfection settings resulting in cell viability of 85% or higher. Knowing the effect of electroporation on cell viability is critical for determining the optimal settings to use for Neon® system transfection.
|Figure 2. GFP and viability display on the Tali® Image Cytometer. Shown is the Tali® cytometer screen after measuring Jurkat cells transfected with the Neon® Transfection System and stained with Tali® Dead Cell Red stain. The assignment of each cell with a particular fluorescence channel is dependent on the threshold set in the fluorescence histogram associated with that channel. After resetting the threshold, the circles in the image on the left side of the screen will update to reflect the new fluorescence levels. The quantitative data, including the percentage of cells expressing GFP and the cell viability, are displayed on the right.|
The final results of this analysis are shown graphically in Figure 3 for all 24 transfection conditions. Each bar represents a Neon® electroporation setting. The green bars represent the percentage of cells positive for GFP fluorescence, as defined by the threshold setting, and refer to the left axis. The red points show the cell viability as measured by Dead Cell Red stain exclusion, and refers to the right axis. In this set of experiments, the settings for well 23 gave the best combination of GFP expression and viability. These settings were 1,500 V and three 10 ms pulses, which agreed well with other experiments on Jurkat cell cultures (1,325 V to 1,500 V and three 10 ms pulses, data not shown). The same optimization protocol was carried out in PC12 and HeLa cells using the Neon® Transfection System and the Tali® Image Cytometer, with specific optimal parameters resulting for each cell type (data not shown).
|Figure 3. Results obtained with the Neon® Transfection System’s optimization protocol combined with analysis with the Tali® Image Cytometer. Jurkat cells were transfected with an EF-1α emGFP plasmid using 24 different combinations of voltage, pulse duration, and number of pulses, using the preloaded optimization settings. The cells were analyzed using the Tali® Image Cytometer, and the percentage of cells expressing GFP as well as the viability of the cells were recorded. The green bars represent percentages of GFP-expressing cells and refer to the left axis; the red points indicate percentages of viable cells and refers to the right axis.|
The Neon® Transfection System, with its preloaded optimization protocol and straightforward workflow, makes testing a variety of electroporation settings quick and easy. The Tali® cytometer provides quantitative GFP expression data, which is not possible with visual inspection of the cells using a standard fluorescence microscope. The results are comparable to what one would get using flow cytometry, but at a fraction of the cost and requiring minimal training. In addition, the Tali® Image Cytometer can be used to measure cell viability, critical information for determining optimal electroporation settings.
Together, the Neon® Transfection System and the Tali® Image Cytometer provide a powerful duo to quickly optimize transfection efficiencies.