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Fluorescence Activated Cell Sorting of Plant Protoplasts
Published on: February 18, 2010
Fluorescence activated cell sorting of plant protoplasts.
Bastiaan O R Bargmann1, Kenneth D Birnbaum
1Department of Biology, New York University, Center for Genomics and Systems Biology, USA.
Journal of Visualized Experiments : Jove
|February 20, 2010
Summary
Fluorescence Activated Cell Sorting (FACS) enables researchers to isolate specific plant cell types for gene expression analysis. This method is practical for obtaining sufficient material, even for rare cells, aiding developmental and stress response studies.
Area of Science:
- Plant molecular biology
- Developmental biology
- Genomics
Background:
- Understanding cell-specific gene expression is crucial for deciphering developmental regulation and environmental responses in multicellular organisms.
- Traditional methods like in situ hybridization offer limited resolution for quantitative analysis, necessitating cell-type-specific RNA isolation for techniques such as quantitative RT-PCR and transcriptome-wide analysis.
- The availability of plant cell type-specific fluorescent reporter lines facilitates targeted cell isolation.
Purpose of the Study:
- To establish and validate a practical method for isolating specific plant cell types using Fluorescence Activated Cell Sorting (FACS).
- To demonstrate the utility of FACS for obtaining sufficient cellular material for high-throughput transcriptome analysis, including rare cell types.
- To present a growth system for Arabidopsis seedlings that allows for controlled treatments prior to cell sorting for stress response studies.
Main Methods:
- Utilized Arabidopsis thaliana root marker lines P(SCR;)::GFP (endodermis, quiescent center) and P(WOX5;)::GFP (quiescent center).
- Dissociated plant tissues into protoplasts via enzymatic digestion and high osmolarity-induced plasmolysis.
- Employed FACS, distinguishing GFP-positive protoplasts by their green vs. red emission spectra under 488 nm laser excitation, and sorted them directly into RNA extraction buffer.
Main Results:
- Demonstrated the straightforward and practical nature of the FACS-based cell isolation technique.
- Successfully isolated sufficient quantities of cells, including scarce quiescent center cells, for transcriptome analysis.
- Presented a seedling growth setup enabling uncomplicated pre-sorting treatments for analyzing biotic and abiotic stress responses.
Conclusions:
- FACS is a viable and efficient method for isolating specific plant cell types for gene expression studies.
- The technique is applicable to scarce cell populations and supports transcriptome-wide analyses.
- The described methodology and growth setup facilitate cell type-specific analyses of plant responses to various stimuli.
