Gene Expression Division, Bio-Rad Laboratories, Inc.
McCoy, A. M., Collins, M. L., Ugozzoli, L. A. Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency. J. Vis. Exp. (35), e1662, doi:10.3791/1662 (2010).
1) Cell preparation
2) Electroporation vessel setup and electroporation
Plate setup and electroporation
Cuvette setup and electroporation
3) Representative Results
After transfecting cells and allowing them to recover, analyze the transfection efficiency qualitatively, using epifluorescent microscopy, and quantitatively, using flow cytometry.
Figure 1. Cells that have been successfully electroporated and are now expressing the GFP gene appear under epifluorescent microscopy.
Figure 2. Viewing the cells under phase contrast allows visualization of both transfected and untransfected cells. These are the cells that were exposed to the lowest voltage electroporation pulse at 200V. The cells are largely confluent due to the high cell density.
Figure 3. The same field of view under epifluorescence shows a number of cells are expressing the GFP marker, but these are only a small percentage of the cells visible in the previous image.
Figure 4. At 250V, the total number of live cells seen under phase contrast decreases slightly.
Figure 5. Under epifluorescence, one can see that the number of GFP expressing cells has increased.
Figure 6. At the highest voltage applied, 375V, there are fewer live cells visible.
Figure 7. However, a large percentage of the remaining cells are expressing GFP. Which condition is optimal depends on the experimental design. In some experiments the largest number of transfected cells might be optimal, in other experiments the highest percentage transfection might be best.
We are interested in the percentage of cells that are GFP positive under each condition and how the percentages vary with cell age. Flow cytometry can provide quantitative information about the transfection results under each of the different electroporation conditions.
Figure 8. Here the percentage of cells that are GFP positive in the passage 5 cells under each of the 12 electroporation conditions are shown. The maximum transfection percentage was approximately 80% under the highest voltage exponential decay pulse, condition 6, and 70% under the strongest square wave pulse tested, condition 12.
Figure 9. With the cells passed 9 times prior to the electroporation, the overall pattern of transfection percentage is nearly identical, but with a very slight decrease in the transfection percentages.
Figure 10. Shown here are the percentages of GFP cells in the passage 13 cells which show a marked decrease in transfection percentage relative to the younger cells. The highest transfection percentages were approximately half what was achieved with the younger cells demonstrating the importance of using healthy cells as soon after isolation as possible.
|Electroporation Conditions used for transfecting MEF cells using the Gene Pulser MXcell|
Exponential Decay pulses,
all with 350 uF, 1000ohm
Square Wave pulses,
all with 2000 uF, 1000 ohm, and 1 pulse
|Voltage (V)||Pulse Duration (ms)|
This video article demonstrates how to use the MXcell electroporation system to easily identify optimal electroporation conditions for MEFs or other primary cell lines. The 96-well plate format allows for many replicates of experimental or optimization conditions to be performed simultaneously, which can eliminate the need for many separate experiments. While carrying out this procedure, one should to remember to use healthy cells as soon after isolation as possible and to use electroporation conditions that are matched to the electroporation buffer.
The authors are employed by Bio-Rad Laboratories that produces reagents and instrument used in this article
|Gene Pulser® Electroporation Buffer||Bio-Rad||165-2676|
|Gene Pulser MXcell™ Electroporation System||Bio-Rad||165-2670|
|Gene Pulser MXcell™ ShockPod™ Cuvette Chamber||Bio-Rad||165-2673|
|Gene Pulser MXcell™ Electroporation System With ShockPod™ Cuvette Chamber||Bio-Rad||165-2674|