Flow cytometry applications: the possibilities are endless

Transcription

1 Flow cytometry applications: the possibilities are endless

2 Flow cytometry applications: the possibilities are endless Antibody staining Surface Staining Intracellular Staining Biological processes Cell Cycle/Cell Proliferation Apoptosis/Cell Death Intracellular processes Gene Expression Fluorescent Proteins (GFP/YFP/etc) Molecular Cytometry Intracellular ions Membranes Plant and Marine Biology Picoplankton and bacteria Cell Isolation Sorting

3 Immunophenotyping Use of labeled antibodies to identify cells of interest Valuable for clinical diagnostics Most common flow assay

4 Intracellular staining Host of intracellular targets Controls critical Non-specific binding can be an issue Different fix/perm strategies have different effects on results FoxP3 marks regulatory T-cells Intracellular marker Multiple antibodies Must validate assay conditions

5 Phospho-Proteins Combining surface and intracellular staining on single cells Advantages Monitor entire signaling cascades Can reveal previously unknown correlations between functions and structures Krutzic and Nolan Cytometry (2003) 55A, 61

6 Cyclins Cyclins appear at specific points in the cell cycle Control Cyclin B DNA Content

7 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

8 Molecular Cytometry Anything that can be tagged with a fluorochrome can be used on a flow cytometer Intracellular ion concentration and ph Cell membrane permeability and membrane potential Membrane fluidity- fluorescence polarization Molecular proximity (FRET)

9 Molecular Cytometry Ca++ with Indo-1 Graf et al., (2007) J Immuno 179:1616

10 Molecular Cytometry FRET pairs can be used to monitor binding of two proteins Measure the 2 nd partner emission from 1 st partner s excitation laser Protein Y Protein X Membrane Excite 10 nm + Emit

11 Molecular Cytometry FRET pairs can be used to monitor binding of two proteins Measure the 2 nd partner emission from 1 st partner s excitation laser Protein Y Protein X Membrane Excite Emit

12 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

13 Marine, Microbiological and Plant applications Aquatic (marine and freshwater) microorganisms characterization Microbiology identification of bacteria, characterization of yeast Genome size and ploidy levels in plants by flow: analysis of homogenates

14 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 90 o -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.

15 Marine, Microbiological and Plant applications Autofluorescence can be useful here to differentiate populations of Cyanobacteria

16 Marine, Microbiological and Plant applications 1m 20m 40m 60m 80m 100m 120m 140m 160m 180m 200m Analysis of aquatic samples using the Influx. Clusters of Prochlorococcus and Synechococcus can be readily distinguished.

17 Marine, Microbiological and Plant applications Bacteria detection by light scatter

18 Marine, Microbiological and Plant applications Bacteria detection by fluorescence (BacLight and PI or SYTOX Green)

19 Marine, Microbiological and Plant applications Bacteria detection by fluorescence (FDA and PI)

20 Marine, Microbiological and Plant applications Bacteria detection by fluorescent proteins

21 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 1 M SYTOX Green or 75 M PI. Analyze with 488nm excitation, triggering on Forward scatter.

22 Marine, Microbiological and Plant applications Flow analysis of Arabidopsis thaliana: Note endoreduplication resulting in multiple peaks of fluorescence.

23 Cell sorting FITC PE

24 Cell sorting The Advantages of flow sorting Pure populations of cells of interest Populations selected on multiple parameters Populations can be selected on level of fluorescence More than one population may be retrieved Single cell sorting The Limitations of flow sorting Cost Expertise required Sorter availability Time taken to sort the required number of cells

25 Cell sorting FL2-H: CD4 PE 100 FL2-H: CD4 PE FL1-H: CD8 FITC FL1-H: CD8 FITC 1000 FL2-H: CD4 PE FL1-H: CD8 FITC FL2-H: CD4 PE 100 FL2-H FL1-H: CD8 FITC FL1-H: CD8 FITC

26 Flow cytometry - It s as easy as this!