Flow cytometry applications: the possibilities are endless
Flow cytometry applications: the possibilities are endless…
Flow cytometry applications: the possibilities are endless… Antibody staining Surface St...
Plant and Marine Biology Picoplankton and bacteria
Cell Isolation Sorting
Immunophenotyping
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Use of labeled antibodies to identify cells of interest Valuable for clinical diagnostics Most common flow assay
Intracellular staining • • • •
Host of intracellular targets Controls critical Non-specific binding can be an issue Different fix/perm strategies have different effects on results
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FoxP3 marks regulatory T-cells Intracellular marker Multiple antibodies – Must validate assay conditions
Phospho-Proteins •
Combining surface and intracellular staining on single cells
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Advantages – Monitor entire signaling cascades – Can reveal previously unknown correlations between functions and structures
Krutzic and Nolan Cytometry (2003) 55A, 61
Cyclins
DNA Content
Cyclin B
Cyclins appear at specific points in the cell cycle
Control
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Gene expression • • • •
Fluorescent proteins from nature Genes well characterized and can be cloned in-frame with gene of interest Can be used to monitor rates of gene expression Useful for cell sorting applications
Molecular Cytometry Anything that can be tagged with a fluorochrome can be used on a flow cytometer
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Intracellular ion concentration and pH
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Cell membrane permeability and membrane potential
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Membrane fluidity- fluorescence polarization
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Molecular proximity (FRET)
Molecular Cytometry Ca++ with Indo-1
Graf et al., (2007) J Immuno 179:1616
Molecular Cytometry • •
FRET pairs can be used to monitor binding of two proteins Measure the 2nd partner emission from 1st partners excitation laser
Protein Y
Protein X
Membrane Excite
10 nm + Emit
Molecular Cytometry • •
FRET pairs can be used to monitor binding of two proteins Measure the 2nd partner emission from 1st partners excitation laser
Protein Y
Protein X
Membrane Excite
Emit
Multiplex Cytometry 100 Color Codes = 100 Simultaneous Tests
Beads are coated with reagents in different ratios and then mixed to make an array
Two dyes are used to create 100 unique colour combinations
Marine, Microbiological and Plant applications
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Aquatic (marine and freshwater) microorganisms – characterization
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Microbiology – identification of bacteria, characterization of yeast
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Genome size and ploidy levels in plants by flow: analysis of homogenates
Marine, Microbiological and Plant applications
Aquatic samples typically contain phytoplankton, cyanobacteria, bacteria, and viruses. Flow cytometry can be used to analyze all of these:
Based on forward angle and 90o-light scatter. Based on endogenous fluorescence (chlorophyll in all phytoplankton, phycobilin pigments in cyanobacteria). Autofluorescence signals can range across six orders of magnitude, making analysis based on autofluorescence challenging. Based on staining with DNA-specific dyes, fluorescence in-situ hybridization (FISH), and fluorophore-labeled monoclonal antibodies.
Marine, Microbiological and Plant applications Autofluorescence can be useful – here to differentiate populations of Cyanobacteria
Marine, Microbiological and Plant applications
1m
60m
140m
20m
80m
160m
40m
100m
120m
180m
200m
Analysis of aquatic samples using the Influx. Clusters of Prochlorococcus and Synechococcus can be readily distinguished.
Marine, Microbiological and Plant applications Bacteria – detection by light scatter
Marine, Microbiological and Plant applications Bacteria – detection by fluorescence (BacLight and PI or SYTOX Green)
Marine, Microbiological and Plant applications Bacteria – detection by fluorescence (FDA and PI)
Marine, Microbiological and Plant applications Bacteria – detection by fluorescent proteins
Marine, Microbiological and Plant applications
Using SYTOX Green and PI: Grow cells to mid-log phase. Fix in cold ethanol. Treat with RNAse and protease. Stain with 1M SYTOX Green or 75 M PI. Analyze with 488nm excitation, triggering on Forward scatter.
Marine, Microbiological and Plant applications
Flow analysis of Arabidopsis thaliana: Note endoreduplication resulting in multiple peaks of fluorescence.
Cell sorting
10000
0.24
FITC
1000
100
10
1 1
10
100 PE
1000
10000
Cell sorting
The Advantages of flow sorting • •
Pure populations of cells of interest Populations selected on multiple parameters
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Populations can be selected on level of fluorescence
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More than one population may be retrieved
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Single cell sorting
The Limitations of flow sorting • • • •
Cost Expertise required Sorter availability Time taken to sort the required number of cells