|
Volume 8 issue 10 October 2007
Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture
Ami A. Deora, Fernando Diaz, Ryan Schreiner, Enrique Rodriguez-Boulan
Supplemental Figure 1: Combined electroporation of basolateral and apical markers. Fully polarized MDCK cells were transfected by electroporation with both CD147-GFP (green) and p75-RFP (red). Both markers are correctly segregated and delivered to the plasma membrane. ZO-1 (white) was immunostained to show integrity of the epithelial monolayer. Shown is the x-z projection of the images obtained by LSCM. Bar: 10µm.
Figure 1 (.jpg)
Supplemental Figure 2: Expression of CD147-GFP in polarized monolayer of Caco-2. Caco-2 cells polarized for 3 weeks were electroporated with CD147-GFP plasmid and expression was assessed after 24 hr. Robust expression of CD147-GFP was seen without affecting integrity of the monolayer. Image was taken with LSCM with 40X objective. Bar: 20µm.
Figure 2 (.jpg)
Supplemental Table 1: Optimization of electrical parameters for efficient electroporation in
Polarized MDCK cells.
High Voltage |
Number of Pulses |
Low Voltage |
Number of Pulses |
Results |
250 |
1 |
25V |
20 |
- |
300 |
1 |
5V |
1 |
- |
|
|
5V |
10 |
- |
|
|
5V |
20 |
- |
300 |
1 |
25V |
1 |
- |
|
1 |
25V |
10 |
+ |
|
1 |
25V |
20 |
+ |
300 |
1 |
50V |
1 |
Monolayer disrupted |
500 |
1 |
5V |
1 |
Monolayer
disrupted |
- : No transfectants; +: Transfected cells observed with intact monolayer
Supplemental Table 2: Determination of transfection efficiency in Caco-2 cells electroporated with CD147-GFP plasmid DNA after 24hr.
Cell line |
Plasmid Concentration (µg/ml) |
Number of GFP positive cells /Total number of cells counted in the fields underneath electrode |
Transfection efficiency (%)
(Mean ± S.D) |
Caco-2 |
5 |
- |
0 |
|
10 |
60/325 |
21.13 ± 5.62 |
|
30 |
90/465 |
19.5 ± 1.45 |
|
