Flow Cytometry Lecture PDF

Flow Cytometry Amr M. Ageez, Ph.D. Yara Ahmed, Ph.D.  Flow Cytometry (FC) Flow cytometry is a standard laser-based technology that is used in the detection and measurement of physical and chemical characteristics of cells or particles in a heterogeneous fluid mixture. The use of flow cy...

Flow Cytometry Amr M. Ageez, Ph.D. Yara Ahmed, Ph.D.  Flow Cytometry (FC) Flow cytometry is a standard laser-based technology that is used in the detection and measurement of physical and chemical characteristics of cells or particles in a heterogeneous fluid mixture. The use of flow cytometry has increased over the years as it provides a rapid analysis of multiple characteristics (both qualitative and quantitative) of the cells. The properties that can be measured by this process include a particle’s size, granularity or internal complexity, and fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that detects the cells based on laser scattered by the cells. A flow cytometer, despite its name, does not necessarily deal with cells; it deals with cells quite often, but it can also deal with chromosomes or molecules or many other particles that can be suspended in a fluid.  Flow Cytometry (FC) Cells are measured individually but in large numbers. Synonymous with FACS (fluorescence-activated cell sorter). Also, simply referred to as “Flow.” “Seeing” Cells Microscopists visualize cells based on their morphology and staining characteristics. Hence, using flow cytometry, a cell can be “seen” both qualitatively and quantitatively. What are the principles in flow cytometry? A technique that enables rapid analysis of the statistically significant number of cells at single cell level. The main principle of this technique is based on scattering of light and emission of fluorescence which occur when a laser beam hits the cells moving in a directed fluid stream. Light scattering by these cells and cellular components occurs due to their structural and morphological properties. Fluorescence is emitted from a fluorescent probe and its intensity is proportional to the amount of fluorescent probe bound to a cell or a subcellular component. Fluorescent probes used in FCM may be fluorochromes linked to antibody molecules or fluorescent dyes that either bind specifically to cellular components or exhibit emission variation depending on some microenvironmental characteristics. Interactions between the cells and the laser beam, in other words, light scattering, and/or fluorescence emission can be observed for each individual cell using specific detectors. Flow Cytometer Instrumentation There are three general components of a flow cytometer: Fluidics Optics Electronics Understanding how a flow cytometer operates is critical to the design and execution of flow cytometry experiments. 1- Fluidics The cell sample is injected into a stream of sheath fluid. By the laminar flow principle, the sample remains in the center of the sheath fluid. The cells in the sample are accelerated and individually pass through a laser beam for interrogation. www.biology.berkeley.edu  2-Optics: When a cell passes through the laser beam, it deflects incident light. Forward-scattered light (FSC) is proportional to the surface area or size of a cell. Side-scattered light (SSC) is proportional to the Introduction to Flow Cytometry: A Learning Guide; Becton Dickinson granularity or internal complexity of a cell. Fluorescence In modern flow cytometers, more than one laser is focused on the sample stream. In this way, not only can cells be measured based on their size and internal complexity, but they can also be measured based on their fluorescent signal intensity. Fluorescence is typically “bestowed” upon a cell through the use of fluorescent dyes called fluorochromes. Fluorochrome Emission The laser beam excites the fluorochrome at a specific wavelength (absorption) and the fluorochrome emits light at a separate wavelength (emission). Note that absorption color differs from emission color. Guide to Flow Cytometry; DakoCytomation 3- Electronics The voltage pulse height, width, and area are determined by the particle’s size, speed, and fluorescence intensity. The pulse parameters are then acquired and analyzed in real-time by a computer. BD LSR II User’s Guide; Becton Dickinson In Addition… Some flow cytometers can sort cells into pre- determined subpopulations. An electrostatic charge is used to deflect a drop containing a fluorescently-labeled cell into one of three collection vessels. www.bio.davidson.edu Differentiating Among Cell Types In the early days of flow cytometry, different cell types were identified based only on their light scattering characteristics. Even though thousands of cells could be rapidly detected, flow cytometry offered little more than what could be achieved by cell counters and microscopy. The introduction of fluorochromes into flow cytometry converted this otherwise limited method of cell detection into a powerful tool for the rapid differentiation of cells. Polyclonal vs. Monoclonal Antibodies Polyclonal antibodies bind to multiple aspects of the same antigen. Their heterogeneity causes problems with standardization when used in flow cytometry. Homogeneous monoclonal antibodies bind to only one aspect of an antigen and will reproducibly label cells. polyclonal antibodies monoclonal antibodies Guide to Flow Cytometry; DakoCytomation Cell-Surface Markers Monoclonal antibodies are used to recognize specific antigens on the surface of cells. These cell-surface markers characterize different cell types. Fluorochrome-tagged monoclonal antibodies brightly label cells for detection by the flow cytometer. Introduction to Flow Cytometry: A Learning Guide; Becton Dickinson  Compensation When the wavelengths of two fluorochromes overlap, the observed fluorescent signal detected by the flow cytometer may not be the actual signal displayed by the cell. In other words, the cell appears to possess a surface marker or phenotype that it does not actually have. Guide to Flow Cytometry; DakoCytomation  Compensation This fluorescence interference can be corrected for by adjusting the measurement parameters of the flow cytometer (either manually or automatically). This correction is termed compensation. In addition, this problem can be avoided by carefully selecting fluorochromes that do not overlap. Guide to Flow Cytometry; DakoCytomation Steps of Flow Cytometry ….1 Sample Preparation Before running in the flow cytometers, the cells under analysis must be in a single-cell suspension. Clumped cultured cells or cells present in solid organs should first be converted into a single cell suspension before the analysis by using enzymatic digestion or mechanical dissociation of the tissue, respectively. It is then followed by mechanical filtration to avoid unwanted instrument clogs and obtain higher quality flow data. The resulting cells are then incubated in test tubes or microtiter plates with unlabeled or fluorescently conjugated antibodies and analyzed through the flow cytometer machine. Jennings, C. & Foon, K.(1997). Blood, 90(8), 2863-2892. Steps of Flow Cytometry ….2 Antibody Staining Once the sample is prepared, the cells are coated with fluorochrome-conjugated antibodies specific for the surface markers present on different cells. This can be done either by direct, indirect, or intracellular staining. In direct staining, cells are incubated with an antibody directly conjugated to a fluorophore. In indirect staining, the fluorophore-conjugated secondary antibody detects the primary antibody The intracellular staining procedure allows direct measurement of antigens presents inside the cell cytoplasm or nucleus. For this, the cells are first made permeable and then are stained with antibodies in the permeabilization buffer. Running Samples At first, control samples are run to adjust the voltages in the detectors. The flow rates in the cytometer are set and the sample Jennings, C. is run.K.(1997). Blood, 90(8), 2863-2892. & Foon, Types of Flow Cytometry 1- Traditional flow cytometers The traditional cytometers are the common cytometer using sheath fluid for focusing the sample stream. 2- Acoustic Focusing Cytometers In these cytometers, ultrasonic waves are used to focus the cells for analysis. This prevents sample clogging and also allows higher sample inputs. 3- Cell sorters Cell sorters are a category of traditional flow cytometers which allows the user to collect samples after processing. 4- Imaging flow cytometer Imaging cytometers are traditional cytometers combined with fluorescence microscopy. Imaging cytometer allows for rapid analysis of a sample for morphology and multi-parameter fluorescence at both a single cell and population level. Jennings, C. & Foon, K.(1997). Blood, 90(8), 2863-2892. Applications of FACS It is used in clinical labs for the detection of malignancy in bodily fluids like leukemia. Cytometers like cell sorters can be used to separate the cells of interest in separate collection tubes physically. It can be used for the detection of the content of DNA by using fluorescent markers. Flow cytometers allow the analysis of replication cells by using fluorescent dye for four different stages of the cell cycle. Acoustic flow cytometers are used in the study of multi-drug resistant bacteria in the blood and other samples. The different stages of cell death, apoptosis, and necrosis can be detected by flow cytometers based on the differences in the morphological and biochemical Jennings, C. & Foon, K.(1997). Blood, 90(8), 2863-2892. changes. Applications - Clinical CD4+ T cell counts are used to monitor the progression of AIDS in HIV-infected patients. www.medic.med.uth.tmc.edu Applications - Research A kinetics assay, such as Ca2+ mobilization, can be performed using a fluorochrome, indo-1, that binds to calcium ions. Cells are loaded with indo-1 and then stimulated to mobilize Ca2+. The UV laser excites the indo-1 and a fluorescent pulse is observed over time. Applications - Research Several fluorochromes (DAPI, propidium iodide, 7-AAD, etc.) bind directly to DNA and are used to estimate the amount of DNA present in a cell. The amount of DNA in a cell determines whether it has entered the cell cycle. www.xenbase.org Cell Sorting Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. Physically separating cells based on some measurable characteristic It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. Fluorescence Activated Cell Sorting Fluorescence Activated Cell Sorting An antibody specific for a particular cell surface protein is associated to a fluorescent molecule and then added to a mixture of cells. For fluorescence when the specific cells pass through a laser beam they are monitored. Droplets containing single cells are given a positive or negative charge, based on whether the cell has limited the fluorescently- tagged antibody or not. Droplets containing a single cell are then detected by an electric field into collection tubes according to their charge. Cell Sorters (FACS – Fluorescence Activated Cell Sorter)  Hydrodynamic Systems Sample in Sheath Piezoelectric crystal oscillator Sheath in Signal Fluorescence direction Sensors Laser beam Scatter Sensor Sheath Core Flow Chamber Limitations of flow cytometry. This process doesn’t provide information on the intracellular location or distribution of proteins. Over time, debris is aggregated, which might result in false results. The pre-treatment associated with sample preparation and staining is a time-consuming process. Flow cytometry is an expensive process that requires highly qualified technicians.