Agarwal, Aparna
(2009)
EMPIRICAL ANALYSIS OF SINGLE-CELL ELECTROPORATION WITH AN ELECTROLYTE-FILLED CAPILLARY.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Electroporation is a technique that uses electric fields to create transient nanopores in a cell's membrane thereby increasing its permeability. In single-cell electroporation, a localized electric field is applied to a single cell to achieve selective permeabilization of the targeted cell without affecting its neighbors. However, there are no experimental reports on the quantitative treatment of single-cell electroporation. We have developed a quantitative approach to control and maximize cell permeabilization and viability in single-cell electroporation. Single-cell electroporation experiments have been performed using small-sized electrolyte-filled capillaries. A549 cells are exposed to the dye Thioglo-1 leading to green fluorescence from intracellular thiol adducts. The fluorescent cells are exposed to brief electric field pulses at varying cell-capillary tip distances. Loss of fluorescence from diffusion of Thioglo-1 conjugates out of the cell is measured as a function of time. Results revealed that longer pulses and a shorter cell-capillary tip distance led to a greater decrease in the cell's fluorescence and are more deadly. A large variability in single-cell electroporation within a set of experimental conditions has been observed. In order to understand the variability in single-cell electroporation, logistic regression has been performed to determine the probabilities of cell survival and electroporation dependence on experimental conditions and cell parameters. The results revealed that the cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio. Further, a quantitative approach has been developed to determine the experimental and cell parameters that influence the outcome of a single-cell electroporation experiment. The regression analysis results revealed that the outcome of electroporation can be related to the cell-capillary tip distance and cell size. The relationship obtained has been used to control the magnitude of molecular flux from single cells, and decrease the variability in the outcome of electroporation of A549, and two more cell lines DU 145, and PC-3 cells. Further, results revealed that cell survivability could be retained across the three cell lines by controlling the magnitude of molecular efflux to 55 % or less.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
5 June 2009 |
| Date Type: |
Completion |
| Defense Date: |
26 March 2009 |
| Approval Date: |
5 June 2009 |
| Submission Date: |
1 April 2009 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
fluorescence; regression; microscopy; statistical analysis |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04012009-153530/, etd-04012009-153530 |
| Date Deposited: |
10 Nov 2011 19:33 |
| Last Modified: |
15 Nov 2016 13:38 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6686 |
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