7. Move the 3D NEP device back to cell incubator for culture.
8. Apply trypsin to release the cells off the 3D NEP chip and
transfer cells back to culture dishes.
3.6 Data Analysis
1. Different voltages are applied to the 3D NEP system keeping
all other parameters (pulse duration/number/interval) constant at 10 ms/5 pulses/1 s.
2. A fluorescence microscope (Nikon Eclipse TI) is used to measure the fluorescence intensity. While acquiring images for the
data analysis, it is highly important to keep all the camera
settings (exposure time, gain, binning, LUTs) the same to be
able to compare the intensity of cells.
3. The intensities significantly increased as the voltages increases.
For example: Electroporating H9C2 cells using FAM-ODNs as
cargo showed an increase in intensity from 50 V to 100 V,
indicating that large amounts of cargo are delivered into the
cells. However, increasing the voltage beyond 140 V did not
show any significant increase in the fluorescent intensity.
4. For dosage controllability, different numbers of pulses are
applied during electroporation keeping other parameters
constant.
5. Individual cells showed an increase in fluorescence intensity as
the pulse number increased, indicating that the electrophoresis
is causing the molecules or cargoes to be delivered into the
cells. Please note that the camera settings should remain the
same while acquiring the images.
6. Fluorescence intensity is measured for each cell using the
acquired images from the fluorescence microscope.
7. Standard deviation of fluorescence intensity is calculated for
both BEP and NEP (see Note 11).
8. An inverted microscope (Nikon Eclipse Ti) is used to image
fluorescent expressions of cells cultured off-chip.
9. Data analysis is performed using commercially available ImageJ
software.
10. Two-sided Student’s T-test is performed to determine the data
significance. Experimental groups with p-values <0.05 are
considered to be significant (see Note 11).
4 Notes
1. Deep reactive ion etching (DRIE) is a process to etch deep
holes or pores in silicon. Specifically, the Bosch process (SF6:
12 s/100 sccm gas flow/700 W ICP power/40 W RF power/
30 mT APC pressure; C4F8: 7 s/100 sccm gas flow/700 W
3D Nanochannel Array for High-Throughput Cell Manipulation and Electroporation
39
8. Apply trypsin to release the cells off the 3D NEP chip and
transfer cells back to culture dishes.
3.6 Data Analysis
1. Different voltages are applied to the 3D NEP system keeping
all other parameters (pulse duration/number/interval) constant at 10 ms/5 pulses/1 s.
2. A fluorescence microscope (Nikon Eclipse TI) is used to measure the fluorescence intensity. While acquiring images for the
data analysis, it is highly important to keep all the camera
settings (exposure time, gain, binning, LUTs) the same to be
able to compare the intensity of cells.
3. The intensities significantly increased as the voltages increases.
For example: Electroporating H9C2 cells using FAM-ODNs as
cargo showed an increase in intensity from 50 V to 100 V,
indicating that large amounts of cargo are delivered into the
cells. However, increasing the voltage beyond 140 V did not
show any significant increase in the fluorescent intensity.
4. For dosage controllability, different numbers of pulses are
applied during electroporation keeping other parameters
constant.
5. Individual cells showed an increase in fluorescence intensity as
the pulse number increased, indicating that the electrophoresis
is causing the molecules or cargoes to be delivered into the
cells. Please note that the camera settings should remain the
same while acquiring the images.
6. Fluorescence intensity is measured for each cell using the
acquired images from the fluorescence microscope.
7. Standard deviation of fluorescence intensity is calculated for
both BEP and NEP (see Note 11).
8. An inverted microscope (Nikon Eclipse Ti) is used to image
fluorescent expressions of cells cultured off-chip.
9. Data analysis is performed using commercially available ImageJ
software.
10. Two-sided Student’s T-test is performed to determine the data
significance. Experimental groups with p-values <0.05 are
considered to be significant (see Note 11).
4 Notes
1. Deep reactive ion etching (DRIE) is a process to etch deep
holes or pores in silicon. Specifically, the Bosch process (SF6:
12 s/100 sccm gas flow/700 W ICP power/40 W RF power/
30 mT APC pressure; C4F8: 7 s/100 sccm gas flow/700 W
3D Nanochannel Array for High-Throughput Cell Manipulation and Electroporation
39