Flow cytometry is a modern immunological technique that uses the laser beam to analyze the physical characteristics of cells suspended in a fluid mixture.
Principle
The fluorescence signal is measured by a detector when light is scattered after interruption by a laser beam as the stained cells flow in a single stream in suspension. The cells have been stained with fluorochrome-tagged monoclonal antibodies specific for the cell markers of interest.
The cells that are stained are excited by the laser then their fluorescence is recorded by a second detector. This second detector is located at an angle of 900 with reference to the laser beam. The intensity of fluorescence reflects the volume/density of the marker of interest
Staining Single Cell suspension for Flow cytometry
For one to use a flow cytometer to analyze cells, such cells should be prepared (Single-cell suspension). The cells are then stained with relevant fluorochrome-labeled monoclonal antibodies. The general protocol is as follows:
The sample cells are harvested from the tissue (e.g. thymus, spleen) and smashed using two surfaces of microscope slides.
The concentration is adjusted to 1-5×106 cells/ml using the standard FACS buffer. This procedure should be done on the ice at 40C. This low temperature and the action of sodium azide which must be added are important because they ensure that the markers of interest are not internalized by the cells. Their internalization would mean a dimmer signal and hence inaccuracy.
The viability of the cells is checked and this viability should be about 95%
About 100 μl of the single-cell suspension should be added to each tube
Blocking antibodies can be added in case there is a high probability of non-specific binding of cells to the Fc receptors with very affinity (e.g. anti- CD16/32). If not, this step can be avoided
An appropriate volume (e.g., 60ul) of labeled primary antibodies is added to well-labeled tubes. For instance, anti-CD4-PE (Tube A), anti-CD8-PE-Cy5 (Tube B), and anti-CD3-FITC (Tube C). If need be, this can be diluted using the FACS buffer. A cocktail is made in an EPPENDORF tube by adding 20ul anti-CD4, 20ul anti-CD8 of 20ul of anti-CD3.
After incubation for 30 minutes, the mixture is washed 3 times. This step is conducted by centrifugation for 5 minutes at 1800rpm.
The mixture is resuspended in FACS buffer at 40C on the ice-cold buffer and stored in a dark place until the planned time for the FACS analysis.
The cells can last for several days without losing their integrity if paraformaldehyde (1-4% ) is added in a case of projected delay in analysis
But for the best results, one should analyze the cells as soon as the preparation of the cell suspension is done.
Flow cytometer mechanics
The mechanical aspect of a flow cytometer includes fluidics, optics, and electronics. Here is a description of each
Fluidics system
This is the channel through which the sample (In a suspension) travels from the sample tube through the flow cell, via the laser beam, via the cell sorter up to waste discard
Optical system
This includes the detector system that records the signal, light sources, optical filters, and lenses. The optical filters help in collecting wavelengths of light in the entire flow cytometer. The different wavelengths are used to analyze different antigens on the cells
The electronics
The electronic system of the flow cytometer has two functions; to convert light signals into voltage or electronic signals, and to carry our data analysis of the sample in question.
To be able to do this, a flow cytometer has two types of photodetectors i.e. the photomultipliers (detects side scatter) and the photodiodes (detects forward scatter).
The detected voltage pulses are measured in quantitative terms by the inbuilt signal processors and recorded in numerical terms in an attached computer system before further analysis is done
Data Analysis and Interpretation
Data analysis for FACs data can be done using the software. The most popular and easy-to-use software for this purpose is Flowjo (Treestar). It will help to analyze different cell populations as they appear on the dot plots.
The dot plots have the x-axis and Y-axis and each represents a specific fluoresce like X-axis may be PE-labelled mAb for a marker while Y-axis may be FITC-labelled mAb for a marker.
The cells being analyzed can be positive for both the markers being analyzed, they can be double negative for the two markers. The dot plot could also show that the cells being analyzed have a single marker either on the X-axis or on the Y-axis.
Depending on what is being analyzed, the scientist interprets the data in the light of theoretical knowledge of what the markers present in some cells and not others may mean.
For example, cells having CD4 and CD3 markers would be T cells and these could be T helper cells or T regs. Analysis of CD25 or FOXP3 would be necessary to differentiate the two types of T cells.
Applications of Flow Cytometry
The flow cytometer is arguably the most important piece of equipment in the field of immunology in modern times. It has found critical use in both research and diagnostic laboratories. In the clinical management of diseases, a flow cytometer is important for the following:
Immunophenotyping
Isolation and quantitation of cells that express a certain marker whose fluorescent monoclonal antibodies are available commercially. It will be possible to give both the absolute number of such cells as well as the percentage of those cells as a proportion of the total number of cells.
An example of such is the total number of cells expressing the CD4 protein marker as a fraction of the total number of white blood cells in an HIV-positive patient. This can be done using a single fluorochrome flow cytometer.
Where there are three markers like CD4, CD8, and CD3 for instance, a three-fluorochrome flow cytometer can be used. Examples of fluorochromes for labeling monoclonal antibodies include FITC (FL1), PE (FL2), and PE-CY5 (FL3).
Cell size
Determination of cell sizes by utilization of light scattering capabilities of the flow cytometer. The information alongside that of distribution of a certain marker/antigen can provide information about the developmental stage of the cells of interest.
This can help in understanding leukemia. The blasts that move out of the bone marrow prematurely are normally bigger in size than the mature cells.
Cells Sub-populations
Differentiate and compare different subpopulations of cells that may be present in a sample using a single analysis. For instance, If you have a fluorescein-tagged monoclonal antibody for a marker expressed by B cells and a phycoerythrin-tagged monoclonal antibody for a marker expressed by T cells, then you can get information on the two cells and compare their populations.
This can be done by the analysis of a sample from the peripheral blood or even the spleen. To know if the cells are functional, they can be analyzed for the presence of TCR (T lymphocytes) and BCR (B lymphocytes) alongside the CD3 markers.
Diagnosis of Cancers
The flow cytometer can be employed in the diagnosis of cancers including Acute lymphoblastic leukemia (ALL) Non-Hodgkin lymphomas, Multiple myeloma, Acute myeloid leukemia, and Chronic lymphocytic leukemia (CLL) using a unique cluster of differentiation markers like CD20 for B cell lymphomas.
Cell cycle analysis
DNA in a cell can be stained using a cell cycle reagent. The DNA binds the dye proportionally based on the amount of DNA in each cell. Using standard modeling algorithms, you can determine cell breakdown in the G0/G1 versus S phase.
You could also study the cells in the G2 phase as well as the polyploid state of the population of cells in your sample. Then you can generate a histogram of cell count versus linear fluorescence to demonstrate the DNA content distribution in all steps of the cell cycle in your sample.
Conclusion
Flow cytometry remains one of the most important techniques in the study of immunology and molecular biology. It is useful in both diagnostic and research laboratories. You can analyze nearly every type of molecule as long as there are monoclonal antibody reagents available.
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