Flow Cytometry Antibodies
Figure 1: Example of immunophenotyping. C57BL/6 murine splenocytes are stained with Anti-Mouse CD3 Monoclonal Antibody (Alexa Fluor 647 Conjugated) (filled gray histogram). Unstained lymphocytes (empty black histogram) are used as control.
What are Flow Cytometry Antibodies?
Flow cytometry antibodies bind to specific antigens on the surface of cells or within cells. They are either directly conjugated with a fluorescent probe, or bind to a secondary antibody that is conjugated to a fluorescent probe. This allows flow cytometers to sort cells based on a particular targeted feature e.g. expression of a specific cell surface receptor. In this way, flow cytometry may be used to count and sort cells, detect changes in protein expression, or assess the cell cycle state of a cell, among other functions.
Popular fluorophore conjugates include:
- Alexa Fluor 488 (AF 488)
- Allophycocyanin (APC)
- Fluorescein isothiocyanate (FITC)
- Phycoerythrin (PE)
- Peridinin-Chlorophyll-protein (PerCP).
Popular Flow Cytometry Antibodies
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Monoclonal vs. Polyclonal Antibodies for Flow Cytometry
Polyclonal antibodies are a mixture of antibodies in which each antibody binds to a different epitope of the same antigen. Polyclonal antibodies are a cost effective way to detect specific cell surface markers by flow cytometry. However, a limitation of polyclonal antibodies is that there is greater background noise. This is because binding is not as specific as with monoclonal antibodies. Monoclonal antibodies are synthesized via the cloning of one hybridoma cell of origin. These type of antibodies bind to the same epitope of an antigen. In this way, there is less non-specific background noise. Therefore, either monoclonal or polyclonal antibodies may be used depending on the requirements of an experiment.
Figure 2: Human peripheral blood lymphocytes are stained with Anti-Human CD197 Monoclonal Antibody (PerCP/Cyanine5.5 Conjugated) and Anti-Human CD3 Monoclonal Antibody (FITC Conjugated).
Flow Cytometry Overview
What is Flow Cytometry?
Flow cytometry is an experimental technique used to analyze both the physical and chemical properties of a population of cells or particles (intracellular proteins, peptides, DNA). Flow cytometers (FC or FCM) utilize laser beam technology to assess the size, shape, and properties of individual cells within a heterogenous population of cells. Flow cytometry is a quantitative method that can be analyzed in depth with the use of specific flow cytometry software programs.
How does Flow Cytometry Work?
Flow cytometry can be used to detect the presence of specific markers expressed either on the surface of the cell or within the cell.
Firstly, a cell suspension sample is prepared and fed into the flow cytometer. By a mechanism known as hydrodynamic focusing, the flow cytometer uses liquid to force the cells to travel at the same speed as single cells. In hydrodynamic focusing, a faster-moving sheath fluid (e.g. saline solution) is used to force the sample into a smaller core stream (referred to as the hydrodynamic core) so that all particles are travelling along the same axis at approximately the same velocity.
Once the sample has been released from the fluidic system it then moves through a laser beam released from the multiplex laser module. The deflected light is then detected by a number of detection systems. By analyzing the output data, one can establish features of the cellular population, such as cell size and complexity, as well as other properties.
Results of Flow Cytometry
When the light from the laser beam passes through a sample there is a deflection of light. This deflected light is sensed by the detectors. This is how the cells within a sample are characterized. For example, if a cell is small in size, light will be deflected at a smaller angle than a bigger cell. One detector measures the scattered light along this path of the laser; this detector is known as the forward scatter detector. The measurement of forward scatter (FSC) allows for the discrimination of cells by size. FSC intensity is proportional to the diameter of the cell, and is primarily due to light diffraction around the cell.
Additionally, the complexity of a cell may be analyzed by flow cytometry. For example, if a cell such as a granulocyte (e.g. neutrophil) passes through the laser beam it will deflect light at a greater angle due to the granularity of the cell. The deflected light in this case is picked up by side scatter (SSC) detectors. Thus, SSC intensity is proportional to the complexity or granularity of the cell.
FSC and SSC data is then combined to characterize the cells/particles in the sample. The analog signal from the detectors is converted to a digital signal, which can then be observed via analysis software, such as FlowJo.
Main Uses of Flow Cytometry
Immunophenotyping/cell sorting by fluorescence - This is one of the most common uses of flow cytometry. Immunophenotyping is the method by which cells, particularly immune cells, are detected and sorted based on their expression of specific antigens e.g. the sorting of T helper cells and cytotoxic T cells based on their expression of CD4 and CD8, respectively.
- Cell sorting by size and complexity - Flow cytometry can be used to analyze the size of cells within a sample (forward scatter), as well and the granularity of cells (side scatter). The granularity of a cell is based on the the internal complexity of the cell i.e. number of organelles. Comparing results with reference forward and side scatter values for specific cell types, one can identify the type of cells within a sample.
- Cell cycle analysis - Flow cytometry may be used to assess what phase of the cell cycle a cell is in using fluorescent dyes which can measure the four distinct phases.
- Cell counting/viability analysis - Viable cells may be distinguished from dead cells using a method known as dye exclusion. This technique works on the premise that dead or dying cells can uptake dye whereas whole, living cells cannot. It is this uptake of the fluorescent dye which allows for the distinguishment of apoptotic and necrotic cells. Additionally, flow cytometry can further differentiate between apoptotic and necrotic cells by analyzing morphological, biochemical and molecular changes within the cells.
- Cell proliferation - Cell proliferation assays are another popular use of flow cytometry. To analyze cell proliferation, the cell membrane of a population of cells is labelled with a fluorescent dye known as carboxyfluorescein succinimidyl ester (CFSE). As mitosis occurs, half of the dye is transmitted to each of the daughter cells. The reduction in fluorescence as a result is proportional to the rate of proliferation.