Flow Cytometry Basics • Flow cytometry is a technique for counting, examining and sorting microscopic particles (usually cells) suspended in a stream of fluid. • Allows simultaneous multi-parametric analysis of the physical and/or chemical characteristics of single cells. • Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis. • Cells from solid tissue must be disaggregated before analysis.
One unique feature of flow cytometry is that it measures fluorescence per cell or particle. This means that the amount of fluorescent signal detected is directly proportional to the number of fluorochrome molecules on the particle. • This contrasts with spectrophotometry in which the percent absorption and transmission of specific wavelengths of light is measured for a bulk volume of sample.
What Can we Measure? • Volume and morphological complexity of cells • DNA and RNA • Proteins • Cell surface antigens (CD markers) • Intracellular antigens (various cytokines, secondary mediators etc.) • Enzymatic activity • pH, intracellular ionized calcium, magnesium, membrane potential • Apoptosis and cell viability • Characterizing multi-drug resistance (MDR) in cancer cells • Plus more!
How it Works • A beam of a single frequency (colour) laser is directed onto a focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam. • one in line with the light beam (Forward Scatter or FSC) • several perpendicular to it (Side Scatter (SSC)) • one or more fluorescence detectors. • Each suspended particle, passing through the beam, scatters the light in some way, and fluorescence chemicals in the particle may be excited into emitting light at a lower frequency than the light source.
By analyzing fluctuations in brightness at each detector it is possible to deduce various facts about the physical and chemical structure of each individual particle (cell). • FSC correlates with the cell volume. • SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
FSC provides a suitable method of detecting particles greater than a given size independent of their fluorescence and is therefore often used in immuno-phenotyping • Correlated measurements of FSC and SSC can allow for differentiation of cell types in a heterogeneous cell population
Lysed Whole Blood Neutrophils Monocytes Lymphocytes
Gating • A subset of data can be defined through a gate. A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis. • For example, in a blood sample containing a mixed population of cells, you might want to restrict your analysis to only the lymphocytes. • Based on FSC or cell size, a gate can be set on the FSC vs. SSC plot to allow analysis only of cells the size of lymphocytes. The resulting display would reflect the fluorescence properties of only the lymphocytes
Fluorescence • An Argon ion laser is commonly used in flow cytometry because the 488-nm light that it emits excites more than one fluorochrome. • Fluorochromes : coloured dyes which accept light energy at a given wavelength and re-emit it at a different wavelength • A simultaneous measurement of different fluorescent dyes is possible, because of similar extinction properties (can be excited by the same wavelength specified by the available laser) and different emission spectras
Fluorochromes • The two main fluorochromes used are fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE) • Two other common fluorochromes are R-PE:cyanine-5 (PE:Cy5) and allophycocyanin (APC)
When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic surface markers of the cell. • In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations. • The staining pattern of each subpopulation, combined with FSC and SSC data, can be used to identify which cells are present in a sample and to count their relative percentages. The cells can also be sorted if desired.
Sorting • In most applications, after a particle exits the laser beam, it is sent to waste. • Sorting allows us to capture and collect cells of interest for further analysis. Once collected, the cells can be analyzed microscopically, biochemically, or functionally
Thanks for your time! • If you have any questions or want more information, please feel free to contact us! www.ucalgary.ca/snyder_chair/contact_info or www.ucalgary.ca/snyder_chair/questions