Contributors:
Sam Livingston, Elkie Fung, Kattie Sepehri, Grace Lin, Jordan Whittier
Technique Chosen:
Flow cytometry
What does this technique do?
- Cell counting, identification, and sorting based on the analysis of laser light scattering from individual cells.
What applications is this technique employed for?
Cell counting: Detect relative population sizes of different cell types within a sample
- Eg. Analyze composition of cells in blood
Cell sorting: physically separating cells or particles of interest from a heterogeneous mixture
- Biomarker detection – Identification of cells based on a specific molecular characteristic (protein, lipid, DNA/RNA etc.)
- Apoptosis detection – identification of cell state by detecting DNA damage, caspase activation, or other apoptotic characteristics
- Protein engineering – rapid sorting of cells expressing desirable proteins
- Cancer detection – identification of many blood cancers by using antibodies that stick to only one type of cell
Cell identification: Identify cells based on external and internal characteristics using light scatter and/or fluorescence (eg. antibody tagging)
What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?
- Are the cells of a shrinking tissue/organs the result of cellular ‘shrinkage’, apoptosis, or necrosis?
- Useful for identifying degenerative conditions and abnormalities in normal organ/tissue homeostasis
- Can the development of cancerous tissue be analyzed through this technique? Useful in identify proteins with improper folding or cells that have passed through stages of the cell cycle which they shouldn’t have.
- Which cells have progressed to a certain cell fate during a specific time period in development?
- How many/which cells of a tissue/organ produce my biomarker of interest?
What critical reagents are required to use this technique?
- A flow cytometer
- fluorescent labels ( eg. specific antibodies or probes)
- solution to suspend cells – usually PBS
- Propidium Iodide for non-specific DNA labelling of apoptotic cells
What critical information is required to be able to employ this technique?
- What particle is being detected and how to tag it (eg. antibody tagging)
- Predicted size of the cell and their surface morphology that can be observed as forward and side scatter by the detectors of the flow cytometer
- Surface properties for tagging/ internal properties for tagging
References
Davies, D. Flow Cytometry. London: Humana Press, 2007. Ebook.
Gasol, Josep M., et al. “Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.”Applied and Environmental Microbiology 65.10 (1999): 4475-4483.
Hodge, Sandra J., et al. “Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease.” Cytometry Part B: Clinical Cytometry 61.1 (2004): 27-34.
Wittrup, K.D. “Protein engineering by cell surface display.” Elsevier 12.4 (2001): 395-392.
Janossy, George, et al. “Affordable CD4+ T cell counts by flow cytometry: II. The use of fixed whole blood in resource-poor settings.” Journal of immunological methods 257.1 (2001): 145-154.
Macey, Marion G. Flow Cytometry. Humana Press Incorporated, 2007.
Sosik, Heidi M., et al. “Growth rates of coastal phytoplankton from time‐series measurements with a submersible flow cytometer.” Limnology and Oceanography 48.5 (2003): 1756-1765.