Fundamentals of Flow Cytometry

From assessing the effects of a drug or experimental treatment to studying and diagnosing pathological conditions or characterizing cell phenotypes, there are many reasons it may be desirable or necessary to analyze cells in the lab. There are a host of parameters that may be assessed and consequently a number of tools commonly found in the cell biology lab. One such important tool is the flow cytometer. This infographic will explore how flow cytometry works, what it can tell us and its applications. How does flow cytometry work? A flow cytometer combines three main systems, fluidics, optics and electronics. When individual cells in the fluidic system pass a laser beam from the optical system, they scatter light, which is then measured by detectors and converted to usable data in the electronic system. What are the applications of flow cytometry? Flow cytometry helps out in many areas of science. Let’s consider some of the common ones. Gating and data analysis Ideally, when using multiple fluorophores, they should be chosen to have minimal spectral overlap, however, some correction is often required. Compensation, utilizing control samples, is used to correct for overlap. Histograms, dot plots and density plots are then generated from the collected data, showing cell populations for each marker. Dot plots show a correlation between two optical values, such as two different fluorescent labels, normally with each cell represented by a dot. The dots representing cells with similar values cluster together and often represent certain cell populations. Assessing cell health and viability No matter what your field of interest or the research question you want to answer, if you are working with live cells, it is important to ensure they are happy and healthy to start with (and are the cells you want) to obtain meaningful and repeatable results. Flow cytometry is therefore a key basic tool in cell culture labs for ongoing checks of cells in use. This can be particularly important, for example, when: • Generating or receiving a new cell line • Cells have been in long-term storage • Cells have been passaged many times • You are isolating cells from tissues or bodily fluids This helps to set a baseline for any subsequent treatments the cells may be subjected to as well. Single-cell analysis Increasingly, studies are addressing features of cells at the single cell rather than bulk level, to understand variations within cell populations and spatial distributions, for which flow cytometry is able to contribute. There has also been work on systems that do not damage or require cells to be labeled to this end. Biopharma and drug discovery Assessing changes in cell health and viability compared to their baseline status can provide valuable information on the effects of drug and biopharmaceutical treatments. Cellular responses such as apoptosis, proliferation and cytotoxicity can be assessed. Flow cytometry has become a vital tool in characterization assays and antibody and phenotypic screening in the discovery and development pipeline, more recently assisted by advances in highthroughput flow cytometry. In the clinic Flow cytometry can assist in diagnosing disease, making prognoses and therapeutic monitoring. In oncology, the technique is used to immunophenotype cells, identifying those with cell surface markers of leukemias or lymphomas, for example, and enabling them to be subclassified. The technique can also be used to identify and separate hematopoietic stem cells that can be used to repopulate bone marrow following chemotherapy for blood cancers. Immunophenotyping can also be used to diagnose primary and secondary immunodeficiency disorders such as X-linked (Bruton’s) agammaglobulinemia and human immunodeficiency virus (HIV) infection respectively. Prior to organ transplant, it is important to ensure a match between organ and donor to minimize the risk of rejection, for which flow cytometry can be employed. Other areas of utility include monitoring cardiovascular disease and sepsis. Microbiology Flow cytometry, while not traditionally part of the microbiology toolkit in part due to cost and sensitivity issues with smaller cells, can be used to identify and quantify microbes without the need for culture. This has been facilitated by advances in acoustic focusing, image enhancement and the development of microbespecific stains in recent years and removes the barriers of some other techniques. Gating is an important step in data analysis. Here, users can draw lines, called gates, to select certain cell populations for further analysis. As a matter of routine, gates are normally included to select for data from single, viable cells. If multiple cells pass the laser at one time, false positives may be generated. Dead cells are also liable to auto fluoresce, which can interfere with results. Additional gates can then be added to focus on specific cell populations of interest. Density plots very similar to dot plots with each cell represented by a dot, can then then be used to identify the densest areas of cell populations and translate this into a color spectrum where red typically shows the highest cell densities and blue the least. Histograms are a type of univariate plot showing the distribution of the number of cells positive for a single optical parameter, such as a specific fluorescent label. What can flow cytometry tell us? There are three main components to flow cytometry output signal, each of which provides information on different aspects of the cell being analyzed. Amplification Optical filters To waste container Fluidic system Optical system Electronic system Detectors Analog-to-digital conversion To computer Excitation laser Cells from sample The fluidic system contains the cells to be analyzed in a buffer or water and is typically pressurized. Before analysis, the cells must be disaggregated and suspended so that they pass through the system one by one. The cells are stained with dyes or fluorescent probes targeted to specific cellular components. 1 The optical system consists of an excitation laser, which is directed at the cells as they pass. The light is scattered by the cells and the resultant light signal is directed by mirrors, filtered for specific wavelengths and recorded by detectors, often photodiodes and photomultiplier tubes (PMTs). Factors such as cell size and complexity influence how the light is scattered. Increases in the number and quality of the lasers and detectors over the years have led to an increase in the number of different markers that can be detected in a single analysis. 2 The electronic system converts the detector signal to a voltage pulse and finally a digital output. This typically includes an amplification step. 3 1 2 3 Forward scatter (FSC) Light continuing forward past the cell → Reflects cell size Light will be bent as it passes the cell, so the greater the FSC signal, the smaller the cell. Side scatter (SSC) Measured at 90° to the path of the light beam → Reflects granularity/complexity of cell Cell structures cause light to be refracted as it passes through the cell, so the greater the SSC signal, the more complex the cell interior. Fluorescence (FL) Light emitted by fluorophores in/on the cell when excited by the laser → Indicates presence of the molecule/structure the fluorophore is targeted to bind. Combined, these metrics can provide information on: Cell viability Cell cycle analysis Cell phenotype Emuneration Proliferation Cell sorting …and help perform tasks such as Inclusion of automated sample loaders is helpful for highthroughput analyses. Fluorescence-activated cell sorters (FACS) can be included to guide different cell types that are positive or negative for a particular parameter into different pots after they are analyzed, rather than a waste container, to allow specific cell populations to be collected. Spectral flow cytometers measure the entire spectrum of a fluorophore rather than just the peak emission, enabling fluorophores with overlapping spectra to be analyzed simultaneously. Imaging cytometry incorporates fluorescence microscopy, enabling cell morphology and other physical changes to be visualized. Forward scatter Side scatter Fluorescent emission Light path Light path Light path ! Relative cell number Fluorescence intensity FITC-conjugated antibody PE-conjugated antibody PE FITC Placebo Drug A One, two, three... Additional instrumentation can be incorporated to tailor the flow cytometer to a specific purpose.