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Assessment of methods and analysis of outcomes for comprehensive optimization of nucleofection.

Bradburne C, Robertson K, Thach D - Genet Vaccines Ther (2009)

Bottom Line: Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70-95% knock down of the gene over 48 h with 90-97% cell viability.Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549.We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Code 6900, 4555 Overlook Ave SW, Washington, DC 20375, USA. bradburne@cbmse.nrl.navy.mil

ABSTRACT

Background: Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line.

Methods: We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated.

Results: GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70-95% knock down of the gene over 48 h with 90-97% cell viability.

Conclusion: Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection.

No MeSH data available.


Related in: MedlinePlus

Heat map of data from the secondary SE optimization. Data has been standardized, with colors indicating high and low values. As seen on the scale, red indicates a high value relative to the mean of the individual data set, while green indicates a low value relative to the mean of the individual data set. Well H2 represents the best combination of GFP fluororescence, cell number, and cell viability.
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Figure 2: Heat map of data from the secondary SE optimization. Data has been standardized, with colors indicating high and low values. As seen on the scale, red indicates a high value relative to the mean of the individual data set, while green indicates a low value relative to the mean of the individual data set. Well H2 represents the best combination of GFP fluororescence, cell number, and cell viability.

Mentions: In order to confirm the initial screening, observe any variation, and evaluate the most promising conditions, a second optimization was performed using the reagent with the best transfection characteristics determined from the screen. Based on the GFP microscopy and fluorescence and the live cell numbers from the initial optimization, reagent SE gave the best overall results of any reagent, and was therefore used in the secondary optimization. The results of the secondary SE optimization are shown in the microscopy images in Figure 1B, the Secondary Optimization data in the supplementary file [see Additional file 1], and the heat map in Figure 2 which allows data to be easily compared and the best wells/conditions to be determined. Similar results were observed, with fluorescence ranging from completely absent to highly fluorescent and cell viability ranging from 68% to 100%. Total live cell number ranged from 1,500 to 134,000, with some wells showing massive cell loss following nucleofection. The optimal condition determined from the secondary nucleofection is in well H2 (Figure 1C) which showed high GFP fluorescence, nominal morphology via microscopy, and a moderate live cell number of 73,000 which falls above the median (45,500). Interestingly, the optimal well determined here differed from that determined in the initial nucleofection, illustrating the utility of a second optimization.


Assessment of methods and analysis of outcomes for comprehensive optimization of nucleofection.

Bradburne C, Robertson K, Thach D - Genet Vaccines Ther (2009)

Heat map of data from the secondary SE optimization. Data has been standardized, with colors indicating high and low values. As seen on the scale, red indicates a high value relative to the mean of the individual data set, while green indicates a low value relative to the mean of the individual data set. Well H2 represents the best combination of GFP fluororescence, cell number, and cell viability.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683797&req=5

Figure 2: Heat map of data from the secondary SE optimization. Data has been standardized, with colors indicating high and low values. As seen on the scale, red indicates a high value relative to the mean of the individual data set, while green indicates a low value relative to the mean of the individual data set. Well H2 represents the best combination of GFP fluororescence, cell number, and cell viability.
Mentions: In order to confirm the initial screening, observe any variation, and evaluate the most promising conditions, a second optimization was performed using the reagent with the best transfection characteristics determined from the screen. Based on the GFP microscopy and fluorescence and the live cell numbers from the initial optimization, reagent SE gave the best overall results of any reagent, and was therefore used in the secondary optimization. The results of the secondary SE optimization are shown in the microscopy images in Figure 1B, the Secondary Optimization data in the supplementary file [see Additional file 1], and the heat map in Figure 2 which allows data to be easily compared and the best wells/conditions to be determined. Similar results were observed, with fluorescence ranging from completely absent to highly fluorescent and cell viability ranging from 68% to 100%. Total live cell number ranged from 1,500 to 134,000, with some wells showing massive cell loss following nucleofection. The optimal condition determined from the secondary nucleofection is in well H2 (Figure 1C) which showed high GFP fluorescence, nominal morphology via microscopy, and a moderate live cell number of 73,000 which falls above the median (45,500). Interestingly, the optimal well determined here differed from that determined in the initial nucleofection, illustrating the utility of a second optimization.

Bottom Line: Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70-95% knock down of the gene over 48 h with 90-97% cell viability.Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549.We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Code 6900, 4555 Overlook Ave SW, Washington, DC 20375, USA. bradburne@cbmse.nrl.navy.mil

ABSTRACT

Background: Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line.

Methods: We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated.

Results: GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70-95% knock down of the gene over 48 h with 90-97% cell viability.

Conclusion: Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection.

No MeSH data available.


Related in: MedlinePlus