Document Detail


Fluorescence activated cell sorting of plant protoplasts.
MedLine Citation:
PMID:  20168296     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.
Authors:
Bastiaan O R Bargmann; Kenneth D Birnbaum
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Video-Audio Media     Date:  2010-02-18
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2010  
Date Detail:
Created Date:  2010-02-19     Completed Date:  2010-03-19     Revised Date:  2014-09-17    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Arabidopsis / cytology*,  genetics
Flow Cytometry / methods*
Gene Expression Profiling / methods
Protoplasts / cytology*,  physiology
Seedling / cytology,  genetics
Grant Support
ID/Acronym/Agency:
5R01GM078279/GM/NIGMS NIH HHS; R01 GM078279/GM/NIGMS NIH HHS; R01 GM078279-05/GM/NIGMS NIH HHS
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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