Looking for tips and tricks on how to improve your flow cytometry experiments? Maybe you're unsure about certain aspects of the technology and need more information? Check out our Flow Post-it program! Born at Memorial Sloan Kettering Cancer Center, it is now a collaborative educational program with the Whitehead Institute for Biomedical Research.
Flow cytometry is a powerful tool, and the ability to gain information in a high throughput manner for samples that are much smaller than “typical” cells is growing in interest. When interested in these small particle experiments, researching extracellular vesicles or viruses for example, it’s important to know the limits of your instrumentation and how to test performance.
Spending a lot of time trying to get your compensation values below 100%? Why?! This post-it details what spillover values over 100% mean and how it impacts your data.
Exposure of cells to laser light in flow cytometers causes molecules such as NADPH & flavins to emit fluorescence. This intrinsic emission of light is what we call autofluorescence (AF). Particles or cells can have varying levels of autofluorescence, and each cell or condition type should have a fully unstained control to account for this.
When analyzing multiparameter flow cytometry data where spillover correction has been carried out, a quality check should always be done to determine the accuracy of results. Visualizing NxN plots with the appropriate scaling to view all events is necessary when doing these quality assessments. Extreme negatives are typically indicative of overcompensation or overestimation of unmixing.
DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain often used to differentiate between live and dead cells for viability measurements in flow cytometry. This reagent is a popular choice due to its short incubation time and high relative brightness. Similar to other reagents, DAPI staining conditions must be optimized for best experimental results.
When designing and executing multiparameter flow cytometry experiments, instrument configuration and panel design both play a very important role. Just because a cytometer can detect fluorochromes does not mean that it will work with your experiment. Care must be taken when designing your panel to avoid fluorochrome combinations that introduce high spread with coexpressing markers.
In order to accurately analyze multicolor flow cytometry data, spectral overlap must be corrected with compensation or unmixing. When running the single stain controls, it is essential that the fluorochromes match those in your experimental samples. Failure to follow this best practice will result in incorrect data.
In multicolor Flow Cytometry experiments, Spillover Spread translates into the width of the positive population in adjacent detectors, and is one of the most important factors impacting resolution. The amount of spillover spread is commonly misunderstood as being a result of compensation or unmixing, but this is not the case. Spreading is a result of photon counting error, prior to any processing of the data such as compensation. The spillover value, on the other hand, is a result of amount of overlap and detector gains, among others. Therefore, independent of spread and has no impact on resolution.
In order to be confident in your sorting results, cell sorter performance must be evaluated through the appropriate QC methods. Rmax is a method that calculates the maximum recovery of the sort sample by looking at how much is lost in the unsorted fraction. Deviations from maximum recovery indicate a problem in sorter performance.
Flow Cytometry commonly utilizes antibodies conjugated to fluorochromes as a means of identifying subsets of cells within a heterogenous sample. Purchasing from a vendor does not guarantee the functionality of the antibody. Validation must be carried out for all antibodies to allow rigor and reproducibility in your Flow Cytometry experiments.
When conjugating two dyes where one fluorochrome’s emission spectra (donor) overlaps the excitation spectra of a second fluorochrome (acceptor) a phenomenon called Förster Resonance Energy Transfer (FRET) occurs, creating a new dye with the excitation maximum of the donor and the emission maxima of the acceptor. The resulting dye is called a Tandem dye.
When carrying out compensation or unmixing in multicolor Flow Cytometry experiments, the use of the appropriate controls is critical. Both beads and cells are often used in these corrections for fluorescence spillover, often times in combination. It is necessary in these cases to have the appropriate autofluorescence referenced for each single color for acquisition and analysis softwares to accurately correct for spillover.
Studying Cell cycle by Flow Cytometry can be performed by staining cells or nuclei with fluorescent DNA markers or nucleoside analogs and measuring the signal output. By using DNA dyes that bind stoichiometrically we can assess DNA ploidy level, cell cycle
stage, the presence of apoptotic cells and performance of drugs for treatment of disease states.
Compensation and unmixing are common methods used to determine the true amount of fluorescence from a fluorochrome when using either traditional or full spectrum Flow Cytometry to carry out multicolor experiments. In these cases, it is necessary to use appro priate single color controls which adhere to all the appropriate rules. If any of these rules are not met compensation or unmixing may not be cor rect. To troubleshoot the acquired panel we need to use a quantitative approach to determine if compensation and unmixing are correctly determined.
Antibody Titration is one of the most important steps in Panel optimization to ensure best Resolution. It allows you to find the optimal concentration of antibody that results in the brightest signal of the positive population while avoiding background staining, thus maximizing signal-to-noise ratio. Additionally, it helps save money and reagents.
Why should we use vital dyes? Distinguishing debris from small cells in tissue preps for Flow Cytometry can often be difficult. Dead cell removal (through nuclear or amine reactive viability dyes) and scatter gating alone cannot be used in scenarios such as these to pull out live cells for analysis or sorting.
In Cancer Research, assessing Cell Proliferation can be useful for a variety of applications, such as cytotoxicity assays, CAR-T cell expansion, drug development, and determining inhibition of tumor growth.
An antibody or immunoglobulin is a Y-shaped protein produced by plasma cells (Fig.1) that serve a variety of immunological functions. The antibody recognizes a specific structure called an antigen (i.e cell surface marker or pathogen), via the unique fragment antigen-binding (Fab) region. The fragment crystallizable (Fc) region can interact with cell surface receptors (Fc receptors) for a variety of biological functions. Fc receptors can be found on subsets such as: B lymphocytes, dendritic cells, monocytes, macrophages, NKs, neutrophils, eosinophils, human platelets, mast cells and basophils.
Cell Viability, autofluorescence and cell aggregation may all affect the quality of cell sorting experiments. Good sample preparation is crucial and will result in better sort purity, yield and post-sort cell function and viability.
Cell Proliferation by Flow Cytometry
Flow Cytometry measures the properties of cells and particles in a stream of fluid, allowing multiparametric analysis at a single-cell level. Fluorescently- labeled cells in suspension are run on flow cytometers where they pass in file, one by one, through one or more lasers of different wavelengths. Scattered laser light or emitted fluorescence are collected and transmitted through optical pathways and amplified/digitized for downstream analysis.