Advanced Flow Cytometry Handbook
Flow cytometry is a powerful, laser-based method for analyzing the physical and chemical characteristics of individual cells in a mixed cell population. Scientists using traditional cytometers will learn how routine workflows can benefit from the speed, throughput, and superior multiplexing offered by the iQue® Advanced Flow Cytometry Platform.
Our intuitive flow cytometers can take you from sample to biologically relevant data faster than any instrument on the market. An elegant and simple program allows you to make sense of complex data sets and accelerate your research.
Whether you're discovering a new therapeutic antibody, developing a specific checkpoint inhibitor or assessing CAR-T cell function, advancing your research depends on the ability to rapidly assess large numbers of samples in a biologically relevant, reproducible, and cost-effective manner.
The selection of appropriate fluorochromes for multiplexing markers of cell phenotype and function in a single well is critical to solving complex cellular research questions to efficiently characterize disease states.
Download the iQue® 3 and iQue® Screener Plus Fluorochrome brochure
FAQ about flow cytometry
Flow cytometry analyzes the physical characteristics of suspension cells and particles using information about their size, complexity (also termed granularity) and relative fluorescence intensity.
The fluidics system of a flow cytometer transports the fluid stream to a laser beam. Cells or particles pass through the laser one by one, in single file. In the optics system component of a flow cytometer, the laser beam illuminates the cells/particles and directs the scattered light and fluorescence to the appropriate detectors.
The electronics system of a flow cytometer converts these light signals into electronic signals that can be processed by your computer. A typical flow cytometer can be set to collect a certain number of events per sample.
Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis. Cells from solid tissue must be desegregated before analysis.
To gather additional information, cells can be labeled with fluorescent molecules. Specifically, fluorochrome-labeled antibodies can be bound to proteins on the cellular surface (antigens). If a cell has many antigens, a large number of fluorochrome-labeled antibodies will bind to it producing a strong fluorescent signal. A cell with no or few antigens will produce a weaker fluorescent signal.
Fluorescent marker, such as a fluorophore-conjugated antibody, directly target an epitope of interest and allow its biological and biochemical properties to be measured. Fluorescent markers are useful in a wide range of applications, including identifying and quantifying distinct populations of cells, cell surface receptors, or intracellular targets, cell sorting, immunophenotyping, and apoptosis studies.
In a flow cytometry experiment, every cell that passes through the interrogation point and is detected will be counted as a distinct event. Each type of light that is detected (forward-scatter, side-scatter, and each different wavelength of fluorescence emission) will also have its own unique channel.
The data for each event is plotted independently to represent the signal intensity of light detected in each channel for every event. This data could be visually represented in multiple different ways. The most common types of data graphs used in flow cytometry include histograms, dot plots and contour diagrams.
Histograms
A histogram is commonly used to compare the fluorescence intensity of two or more populations.
Dot Plots
Dot plots compare 2 or 3 parameters simultaneously on a scatter-plot where each event is represented as a single point (or dot). The dot plot is a figure that shows the relationship between multiple variables at once, and the parameters can be any combination of scatter and fluorescence signals.
Contour Plots
Contour plots display the relative frequency of the populations, regardless of the number of events collected. A contour diagram displays the probability contouring with joined lines representing similar numbers of cells. Concentric rings form around populations so that the higher the density, the closer the rings are on the contour diagram.
Figure 1. Examples of flow cytometry data. (A) Raji cells (brightly labeled with iQue® Cell Proliferation and Encoding (V/Blue) Dye), Ramos cells (dimly labeled with encoder dye) and Jurkat cells (unlabeled) were combined (5K/well) for a CDC assay and separated based on their encoder dye fluorescence, as displayed in the histogram. (B) Dot plot example of pre-defined gates on iQue Forecyt® enable automatic phenotyping of human T cell subsets. (C) Contour plot with clear gating of the CD14 positive population.
Flow cytometry is a powerful tool that has applications in immunology, molecular biology, bacteriology, virology, cancer biology and infectious disease monitoring. The most used application in flow cytometry is immunophenotyping. It utilizes the unique ability of flow cytometry to simultaneously analyze mixed populations of cells for multiple parameters such as surface markers, cytokine analysis and cell health.
