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Improvement of the fluorescence intensity during a flow cytometric analysis for rice protoplasts by localization of a green fluorescent protein into chloroplasts.

You MK, Lim SH, Kim MJ, Jeong YS, Lee MG, Ha SH - Int J Mol Sci (2014)

Bottom Line: Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals.From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency.Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population.

View Article: PubMed Central - PubMed

Affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Korea. minkyou@khu.ac.kr.

ABSTRACT
Protoplasts have been a useful unicellular system for various molecular biological analyses based on transient expression and single cell analysis using fluorescence-activated cell sorting (FACS), widely used as a powerful method in functional genomics. Despite the versatility of these methods, some limits based on low fluorescence intensity of a flow cytometric analysis (FCA) using protoplasts have been reported. In this study, the chloroplast targeting of fluorescent proteins (FPs) led to an eight-fold increase in fluorescence intensity and a 4.5-fold increase of transfection ratio from 14.7% to 65.7% as compared with their targeting into the cytoplasm. Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals. To investigate the reason underlying this finding, mRNA/protein levels and transfection efficiency were analyzed, and results suggest that mRNA/protein levels and transfection ratio are not much different between K-sGFP and KR-sGFP. From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency. Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population. Our study could be usefully applicable for highly sensitive FACS and FCA-investigations of green tissue.

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Comparative analysis of FCA using rice protoplasts among sGFP, K-sGFP and KR-sGFP constructs. Rice protoplasts were transfected with 5 μg plasmids of sGFP, K-sGFP, and KR-sGFP constructs. The FCAs were performed using single live cells gated by the side/forward scattering. (A) The fluorescence intensities were analyzed using the fluorescein isothiocyanate (FITC-A) channel of the flow cytometer and normalized by the average fluorescence intensity of the K-sGFP construct. The results are presented in arbitrary units; (B) The ratio of protoplasts with a fluorescence intensity exceeding the threshold value (103) to the gated protoplasts was calculated on FCA. The left axis shows the percentage of the transfected protoplasts expressing sGFP and the right axis shows their arbitrary results. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA and (C) Flow cytometry histograms (left panels) and the images of hemocytometer measurements (right panels). The fluorescence intensity on FITC was partitioned into P1 (103–104), P2 (104–105), and P3 (>105) subpopulations, and M means “the missing population”. For a hemocytometer analysis, 10 μL of the transformed protoplasts was mounted on a hemocytometer, and were photographed using the DIC and FITC-A channels of a confocal microscope (200× magnification). The merged images of one square (1 mm2) on the hemocytometer were shown. The sGFP-expressing cells were identified as the green fluorescent signals, and the yellow scale bars on the images show the mean intensity ratio (for 106 pixels) of the green fluorescence, analyzed by a Histogram tool.
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ijms-16-00788-f002: Comparative analysis of FCA using rice protoplasts among sGFP, K-sGFP and KR-sGFP constructs. Rice protoplasts were transfected with 5 μg plasmids of sGFP, K-sGFP, and KR-sGFP constructs. The FCAs were performed using single live cells gated by the side/forward scattering. (A) The fluorescence intensities were analyzed using the fluorescein isothiocyanate (FITC-A) channel of the flow cytometer and normalized by the average fluorescence intensity of the K-sGFP construct. The results are presented in arbitrary units; (B) The ratio of protoplasts with a fluorescence intensity exceeding the threshold value (103) to the gated protoplasts was calculated on FCA. The left axis shows the percentage of the transfected protoplasts expressing sGFP and the right axis shows their arbitrary results. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA and (C) Flow cytometry histograms (left panels) and the images of hemocytometer measurements (right panels). The fluorescence intensity on FITC was partitioned into P1 (103–104), P2 (104–105), and P3 (>105) subpopulations, and M means “the missing population”. For a hemocytometer analysis, 10 μL of the transformed protoplasts was mounted on a hemocytometer, and were photographed using the DIC and FITC-A channels of a confocal microscope (200× magnification). The merged images of one square (1 mm2) on the hemocytometer were shown. The sGFP-expressing cells were identified as the green fluorescent signals, and the yellow scale bars on the images show the mean intensity ratio (for 106 pixels) of the green fluorescence, analyzed by a Histogram tool.

Mentions: To determine the effect on a FCA according to each different subcellular localization of sGFPs, the rice protoplasts transfected with each of cytoplasm (sGFP or K-sGFP) or chloroplast (KR-sGFP)-targeting construct were split into four tubes to perform four experiments: (i) a flow cytometric analysis; (ii) quantitative real-time PCR; (iii) western blot analysis; and (iv) hemocytometer measurement. Each analysis was repeated in three independent experiments (Figure 2 and Figure 4).


Improvement of the fluorescence intensity during a flow cytometric analysis for rice protoplasts by localization of a green fluorescent protein into chloroplasts.

You MK, Lim SH, Kim MJ, Jeong YS, Lee MG, Ha SH - Int J Mol Sci (2014)

Comparative analysis of FCA using rice protoplasts among sGFP, K-sGFP and KR-sGFP constructs. Rice protoplasts were transfected with 5 μg plasmids of sGFP, K-sGFP, and KR-sGFP constructs. The FCAs were performed using single live cells gated by the side/forward scattering. (A) The fluorescence intensities were analyzed using the fluorescein isothiocyanate (FITC-A) channel of the flow cytometer and normalized by the average fluorescence intensity of the K-sGFP construct. The results are presented in arbitrary units; (B) The ratio of protoplasts with a fluorescence intensity exceeding the threshold value (103) to the gated protoplasts was calculated on FCA. The left axis shows the percentage of the transfected protoplasts expressing sGFP and the right axis shows their arbitrary results. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA and (C) Flow cytometry histograms (left panels) and the images of hemocytometer measurements (right panels). The fluorescence intensity on FITC was partitioned into P1 (103–104), P2 (104–105), and P3 (>105) subpopulations, and M means “the missing population”. For a hemocytometer analysis, 10 μL of the transformed protoplasts was mounted on a hemocytometer, and were photographed using the DIC and FITC-A channels of a confocal microscope (200× magnification). The merged images of one square (1 mm2) on the hemocytometer were shown. The sGFP-expressing cells were identified as the green fluorescent signals, and the yellow scale bars on the images show the mean intensity ratio (for 106 pixels) of the green fluorescence, analyzed by a Histogram tool.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4307275&req=5

ijms-16-00788-f002: Comparative analysis of FCA using rice protoplasts among sGFP, K-sGFP and KR-sGFP constructs. Rice protoplasts were transfected with 5 μg plasmids of sGFP, K-sGFP, and KR-sGFP constructs. The FCAs were performed using single live cells gated by the side/forward scattering. (A) The fluorescence intensities were analyzed using the fluorescein isothiocyanate (FITC-A) channel of the flow cytometer and normalized by the average fluorescence intensity of the K-sGFP construct. The results are presented in arbitrary units; (B) The ratio of protoplasts with a fluorescence intensity exceeding the threshold value (103) to the gated protoplasts was calculated on FCA. The left axis shows the percentage of the transfected protoplasts expressing sGFP and the right axis shows their arbitrary results. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA and (C) Flow cytometry histograms (left panels) and the images of hemocytometer measurements (right panels). The fluorescence intensity on FITC was partitioned into P1 (103–104), P2 (104–105), and P3 (>105) subpopulations, and M means “the missing population”. For a hemocytometer analysis, 10 μL of the transformed protoplasts was mounted on a hemocytometer, and were photographed using the DIC and FITC-A channels of a confocal microscope (200× magnification). The merged images of one square (1 mm2) on the hemocytometer were shown. The sGFP-expressing cells were identified as the green fluorescent signals, and the yellow scale bars on the images show the mean intensity ratio (for 106 pixels) of the green fluorescence, analyzed by a Histogram tool.
Mentions: To determine the effect on a FCA according to each different subcellular localization of sGFPs, the rice protoplasts transfected with each of cytoplasm (sGFP or K-sGFP) or chloroplast (KR-sGFP)-targeting construct were split into four tubes to perform four experiments: (i) a flow cytometric analysis; (ii) quantitative real-time PCR; (iii) western blot analysis; and (iv) hemocytometer measurement. Each analysis was repeated in three independent experiments (Figure 2 and Figure 4).

Bottom Line: Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals.From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency.Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population.

View Article: PubMed Central - PubMed

Affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Korea. minkyou@khu.ac.kr.

ABSTRACT
Protoplasts have been a useful unicellular system for various molecular biological analyses based on transient expression and single cell analysis using fluorescence-activated cell sorting (FACS), widely used as a powerful method in functional genomics. Despite the versatility of these methods, some limits based on low fluorescence intensity of a flow cytometric analysis (FCA) using protoplasts have been reported. In this study, the chloroplast targeting of fluorescent proteins (FPs) led to an eight-fold increase in fluorescence intensity and a 4.5-fold increase of transfection ratio from 14.7% to 65.7% as compared with their targeting into the cytoplasm. Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals. To investigate the reason underlying this finding, mRNA/protein levels and transfection efficiency were analyzed, and results suggest that mRNA/protein levels and transfection ratio are not much different between K-sGFP and KR-sGFP. From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency. Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population. Our study could be usefully applicable for highly sensitive FACS and FCA-investigations of green tissue.

Show MeSH
Related in: MedlinePlus