How it Works
How Flow Cytometry Works
A flow cytometer is made up of three main components: fluidics, optics and electronics.
In order for a researcher or a clinician to analyze one cell at a time, the sample (e.g., human blood) must be transported in a single stream, such that each cell independently passes through a laser beam for interrogation. The fluidics system is responsible for introducing the funneled sample into the flow cytometer.
As each cell independently passes through the laser beam, it scatters light based on a number of different physical characteristics. The optics system is responsible for collecting the scattered light, which is then passed on to various detectors.
In order to be able to visually analyze data generated through flow cytometry, scattered light signals passed from the optics system to the detectors must be converted into numerical values. The electronics system is responsible for this data conversion from light to numbers.
Light Scatter and Fluorescence
As each cell passes through the laser beam at the interrogation point, the laser light is scattered. This scattering is dependent on a number of physical properties of the cell, such as size and internal complexity.
Forward-scattered light (FSC) is directly proportional to cell-surface area or size of the cell. FSC can be used to detect particles that are greater than a given size independent of their fluorescence and is often used in immunophenotyping.
Side-scattered light (SSC) is proportional to internal complexity.
Fluorescence is the emission of light by a substance that has absorbed light. A fluorescent compound absorbs light energy over a range of wavelengths that is characteristic for that compound. The absorption of light causes an electron to be excited to a higher energy level; the excited electron quickly decays to its ground state and the excess energy is emitted as a photon of light. This transition of energy is called fluorescence. The Absorption Spectrum is the range over which a fluorescent compound can be excited. The Emission Spectrum is the range of emitted wavelengths for a particular compound.
Monoclonal antibodies allow researchers to detect and label specific populations of cells. When multiple cells and parameters are studied, it is critical to have a way to identify and group cells. To achieve this, the use of fluorescence and multicolor staining was incorporated into flow cytometry.
Linking monoclonal antibodies with fluorochromes allows researchers to track the cells that bound to the antibody. Because most cells do not naturally emit fluorescent light, the fluorochrome-tagged monoclonal antibody light signal will be picked up with that cell passes through the interrogation point.
Most fluorochromes differ from each other with respect to the color of light that they will emit. It is important to verify that all fluorochrome-antibody conjugates used in a given experiment do emit different colors so that different populations of cells can be distinguished from one another.
Importance of Controls
Controls are absolutely essential to flow cytometry and the following controls should be prepared for every experiment.
A tube of cells with no fluorochromes added with the exception of a viability dye. The cells should be subjected to all the resuspensions and washes that your stained samples require. This control is used to set the voltage level for the scatter and fluorescence channels.
Positive control tubes, each stained with a single color for every fluorochrome used. These act as your compensation controls and are used to measure the spillover between fluorescence detection channels (which produces false positive signal). Spillover is dependent on the setup of the machine for that day, not the experiment, so you cannot simply use the compensation settings from your last experiment — it must be calculated each and every time.
Fluorescence Minus One (FMO) Controls
When staining with a large number of colors (i.e., more than three), you may see cells that look like they have stained positive, but are in fact artifacts, due to spillover between fluorescence channels, revealed by applying compensation to the data. For this reason it is useful to run controls, which are stained for all of the colors in your assay except one. This will allow you to set the boundary between negative and positive staining with certainty.
A. Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.
A. There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.
A. Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the auto fluorescence associated with any particular cell type.
A. Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.
A. Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal "bleeds" into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.
A. Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.
A. Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET).
A. I would always recommend performing a titration because sometimes you find you can use less antibody than what is recommended for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands. The % positive staining should be maintained with concentration selected - i.e. if while they are titrating they find the population % drops then they are not staining all the cells and should use a higher concentration.
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