Basic Flow Cytometry (FCM) /FACS Primer:
1. Air pressure is used to drive cells through a FACS machine.
2. 'Sheath fluid,' basically isotonic buffered water, dilutes the cell suspension inside the machine.
3. The cells, present as a suspension in an isotonic buffer, ultimately move past a laser and electronic detector system in a single file.
4. Different FACS machines may be equipped with lasers that emit lights of different wavelengths.
5. As the cells move past the laser, they reflect and refract the light from the lasers, and they also glow or fluoresce, depending on the nature of fluoresce on or in the cells.
6. Various detectors pick up such signals from the cells. These 'signals' are also referred to as 'parameters'.
7. The FSC or forward scatter parameter indicates the light reflected back by a cell. In general, it is a measure of the cell size Larger cells have bigger FSC values, while cell debris and dying cells (generally speaking) have low FSC values.
8. The SSC or side scatter parameter indicates the light refracted by the cell at an angle to the incident laser light Cells that are more granular a are dying (generally speaking) have higher SSC values.
9. A cells, besides reflecting light, will fluoresce if it has fluorochromes that are excitable by light of particular wavelengths emitted by the lasers. The wavelength of this fluorescent light is always higher than that of the incident laser light It varies depending on the nature of the fluorochrome.
10. For example. FITC fluorochrome fluoresces green while PE fluorochrome fluoresces orange.
11. FACS machines have different detectors for such different 'colors.' Thus, a cell's FL1, FL2, FL3, etc, parameters are measured.
12. Each sample analysis thus involves the measurement of all these parameters for a specified number of cells (usually, 10,000) The data recorded look something like this:
13. In the end, a software application is used to plot the values as histograms, dot-plots, etc.
14. Fluorochromes do not emit light of just one wavelength It is instead a range of wavelengths Similarly, the electronic detectors do not detect light of just one wavelength, but a range of wavelengths Thus, light may 'leak' from and get detected by more than one detector.
15. TNis is important to consider and 'compensate' for if one is detecting multiple fluorochromes on or in the same cell.
16. Fluorochromes in a cell can be of various types For example, a cell might be expressing a green fluorescent protein such as GFP. A cell's DNA might be stained with a fluorochrome such as propidium iodide. Or. antibodies, either with fluorochrome chemical tags or 'tagged' with 'secondary antibodies' with such tags, might be bound to antigen-bearing molecules in or on the cell.
Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells.
It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA.
The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with unlabeled or fluorochrome-labeled antibodies and analyzed on the flow cytometer.
• Unlabeled cells as blank control
• Single color and FMO control
• Isotype control
• Negative cells control
• Excitation and Emission
• Fluorochrome Selection
• Sample preparation
• Fluorescence staining
• What fluorochromes can I use?
• What controls do I need?
• Do the cells need to be permeabilized?
• Do the samples need to be fixed?
• Introduction to Flow Cytometry
• Flow Cytometry Basic Training
• Principles and Application of Flow Cytometry
• No Staining
• Weak Staining
• Nonspecific Staining
• Unexpected Staining