Flow cytometry is a complex field that draws people from diverse scientific backgrounds. Whether you’re an immunologist or aquatic ecologist, researcher or clinician, we hope this guidebook will help you in your journey of discovering the powerful technology and applications of flow cytometry.
Flow cytometry is the science of measuring physical and chemical properties of live cells or other biological particles as they pass in a fluid, single-cell stream through a measuring apparatus. In the most common scenario, one or more lasers interrogate each particle and, at a minimum, the system measures the degree and direction of scattered light — indicators of the particle’s size, shape and structure. If particles have been stained with one or more fluorescent dyes — known as fluorochromes — the light source excites these dyes to provide additional biological information about each particle, such as metabolic activity, DNA content and the presence of specific surface and intracellular markers. Precise optical and electronic elements collect the fluorescent pulses and scattered light, convert them into digital values and send them to a computer for analysis. Some flow cytometers are also equipped to identify and sort user-specified particles into collection vessels. High-performance cell sorters can routinely reach rates of 70,000 cells per second.
The unique power of flow cytometers is that they can rapidly and quantitatively measure multiple simultaneous parameters on individual live cells and then isolate cells of interest. Additionally, the sensitivity and throughput rates achievable by high-performance commercial instruments enable detection of extremely rare populations and events (frequencies below 10-6), such as stem cells, dendritic cells, antigen-specific T cells and genetic transfectants.1 As a result, applications for flow cytometers continue to grow.
In addition to traditional immunology and pathology applications involving particles such as lymphocytes, macrophages, monocytes and tumor cells, flow cytometers are widely used in conjunction with fluorescence-based protein reporters, such as green fluorescent protein (GFP). In this arena, flow cytometers can monitor both transfection efficiency and protein expression levels. They also can detect fluorescence resonance energy transfer (FRET), which provides information about molecular interactions, protein structure and DNA sequence .
Ongoing development efforts in the flow cytometry industry are aimed at automation and laboratory integration. Input/output robotics, pushbutton operation and automated sample preparation will increase throughput rates and make the technology more accessible to a wider user base, as new fluorescent dyes and creative screening approaches expand applications into the proteomic arena. Eventually, software advances will seamlessly network instruments into comprehensive analytical and diagnostic systems, and the industry may marry its technology with imaging and microfluidics. Obviously, this is only a very superficial discussion of the field of flow cytometry. It is intended merely to set the stage for the rest of the publication, which will introduce you to more details about the technology and applications of flow cytometry.
How Does Flow Cytometry Work?
The basic principle of flow cytometry involves the passage of cells in a stream through a laser beam. The cellular particles could be fluorescently labeled and then excited by the laser to emit light at varying wavelengths. Each cell passing through the cytometer scatter some of the laser light and emit fluorescence following the excitation. The instrument then measures the fluorescence as well as the scattered light to determine the amount and type of cells present in the population.
Several parameters are simultaneously measured by a flow cytometer;
- Forward scatter intensity (FSC): FSC is directly proportional to the cell diameter.
- Side scatter intensity (SSC): SSC represents the granularity of the cell.
- Fluorescence intensity: Light emitted at various wavelengths is measured.
FSC alone is quite useful in excluding cell debris, cell aggregates, and dead cells. It is sufficient in distinguishing lymphocytes from granulocytes or monocytes in blood samples. The granularity of living cells like dendritic cells can be determined from SSC. Fluorophores coupled to antibodies are used to mark specific subpopulation of cells, and thus to quantify, sort, or study those cells from a heterogeneous cell population.
The ability of flow cytometers to measure the flourescense has many more applications in biology.
Applications of Flow Cytometry:
The research applications of flow cytometry include:
– Immunophenotyping: Flow cytometry is most commonly used in immunophenotyping, which detects and quantifies a specific population of cells in a heterogeneous sample, usually lymph, blood, or bone marrow. Hematological malignancies like leukemia and lymphomas can be diagnosed in clinical labs by immunophenotyping the sample.
– Transfection efficiency: By using fluorescent protein (i.e. GFP) as a marker, flow cytometry can be used to determine the transfection efficiency. The flow cytometer can determine the percentage of successfully transfected cells within a transfected cell population.
– Apoptosis: Apoptosis, the programmed cell death is a normal process among the eukaryotic cells. Cells can die due to a variety of reasons and apoptosis in cells can be detected by several flow cytometric methods. Cells are usually stained with 7ADD or Annexin V to determine apoptosis.
– Cell cycle analysis: By fixing the cells and then staining the cellular DNA with dyes like ethidium bromide, propidium iodide, and DAPI, flow cytometry can be used to quantify the cells in G1, S, G2 and M phases. Since the dye binds to DNA, the fluorescent signal is directly proportional to the amount of DNA present inside the cell, which varies with different phases of the cell cycle.
– Cell proliferation: Cell proliferation is often studied in cell biology to measure cellular metabolic activity in response to stimuli like cytokines, media components, and growth factors. By labeling the cells with carboxyfluorescein succinimidyl ester (CFSE), a cell membrane fluorescent dye, the flow cytometer can measure the proliferation of cells. As the cell divides, the dye is passed on to the daughter cells, each getting half the original dye. Researchers can calculate the proliferation by measuring the reduction in fluorescence signal.
– Cell sorting: A particular subset of cells can be sorted from a heterogeneous population based on desired parameters.
– Membrane potential: DiOC2 is a fluorescent membrane-potential indicator dye which exhibits green fluorescence in all bacterial cells. In cells with larger membrane potentials, the dye becomes more concentrated and shifts to red. The green or red bacterial populations can be easily distinguished using a flow cytometer.
– Live/dead bacteria discrimination: Using the combination of two dyes thiazole orange and propidium iodide, the flow cytometer provides a reliable method to discriminate live and dead bacteria. The efficacy of antibiotics in killing microbes can be determined this way.
While this was just a glimpse, Dive Into The Course To Learn More In Detail.