Flow Cytometry
Bio-Rad is a leading supplier of antibodies, reagents, and instruments for flow cytometry. To help you with your experiments, we provide practical advice on all aspects of flow cytometry from setup to analysis giving you confidence in your results.
Flow Guide
Introduction
This flow cytometry guide gives you a basic overview of all the important features of flow cytometry. Whether you are new to flow cytometry, learning new techniques, wanting to build better panels or just refreshing your knowledge, this guide can help you. We have included a flow cytometry glossary of terms and created a handy pocket size version for quick reference.
Gating Strategies
Flow cytometry data analysis is built upon the principle of gating. Gates and regions are placed around populations of cells with common characteristics - usually forward scatter, side scatter and marker expression - to investigate and quantify these populations of interest. Here we show what the common flow cytometry graph outputs look like and how in a few simple steps you can identify different cell populations that have been stained with antibodies conjugated to fluorophores.
Data Analysis
Flow cytometry data can be analyzed in different ways. Here we give common examples of gating strategies and how data can be best represented:
Forward and side scatter
The first step in gating is often distinguishing populations of cells based on their forward and side scatter properties. Forward and side scatter can give an estimation of the size and granularity of the cells respectively, although this can depend on several factors such as sample, wavelength of the laser, collection angle and refractive index of the sample and sheath fluid. The red/yellow/ green/blue hot spots indicate increasing numbers of events resulting from discrete populations of cells. Debris often has a lower level of forward scatter and is found at the bottom left corner of the density plot. The forward scatter threshold can be increased to avoid collecting these events, or they can be removed by gating on the populations of interest, shown by the red gates in figure 1b and 1c.
Single parameter histograms
The events or cells within a gate can be further analyzed for expression of markers by fluorescence and the data expressed in a histogram. In order to accurately identify the positive dataset, flow cytometry should be repeated in the presence of appropriate controls. This is particularly necessary if a single distinct peak is observed, however often in flow cytometry multiple peaks are observed due to mixed populations as can be seen in Figure 2, where CD4 expression in analyzed.
Two-parameter density plots
These graphs display two measurement parameters, one on the x-axis and one of the y-axis and the events as a density (or dot) plot...
Backgating
Backgating is a useful method of identification of cells to confirm a staining pattern or gating method...
Controls
Controls are vital in any experiment to reliably distinguish your results from background variation and non-specific effects. Here we will discuss some essential controls for flow cytometry you must consider to ensure publication quality flow cytometry data.
Controls in Flow Cytometry
Controls are vital in any experiment to reliably distinguish your results from background variation and non-specific effects. Here we will discuss some essential controls for flow cytometry you must consider to ensure publication quality flow cytometry data:
Unstained controls
The first thing to identify in flow cytometry is your cell population by its forward and side scatter characteristics. After this, you need to determine where the negative population will be. To do this you should always have an unstained population. This will allow you to determine the level of background fluorescence or autofluorescence (Figure 1b) and set your voltages and negative gates appropriately to allow you to visualize positive populations (Figure 1e).
Single staining and compensation controls
When performing multicolor flow cytometry, single stained samples are essential to determine the levels of compensation...
Viability controls
The presence of dead cells in your sample can greatly affect your staining and therefore the quality of your data...
Fc blocking controls
Fc receptors are found on monocytes, macrophages, dendritic cells and B cells. As the name suggests they bind antibodies via their constant Fc domain rather than the antigen specific Fab domain...
Fluorescence minus one controls
Fluorescence minus one (FMO) controls are important when building multicolor flow cytometry panels as they will help you determine where your gates should be set...
Tube |
FITC |
PE |
PE-A750 |
PE-Cy5 |
|---|---|---|---|---|
|
Unstained |
- |
- |
- |
- |
|
FITC – FMO |
- |
+ |
+ |
+ |
|
PE - FMO |
+ |
- |
+ |
+ |
|
PE-A750 - FMO |
+ |
+ |
- |
+ |
|
PE-Cy5 - FMO |
+ |
+ |
+ |
- |
Intracellular staining controls
Isotype controls have been optimized for cell surface staining to control for non-specific binding of antibody and fluorophore...
Biological controls
In addition to staining and isotype controls, you should also consider biological controls that will enable you to determine staining specificity and experimental limitations...
StarBright Dyes
StarBright Dyes are fluorescent nanoparticles, specifically developed for flow cytometry. These proprietary fluorescent dyes are bright, photostable, fixable, and can be used in any buffer with no loss of performance. They have been developed to have narrow excitation and emission profiles for multicolor panel building in conventional flow cytometry, but also have unique spectra for use in full spectrum flow cytometry.
Find out how to build bigger brighter panels with StarBright Dyes.
Figure 1. Excitation and emission profiles of StarBright Blue and StarBright Violet Dyes.
More Resources
Common Protocols
Whilst it is not possible to describe every method, the flow cytometry protocols below provide detailed procedures for the common treatment and staining of cells prior to acquiring on a flow cytometer.
There can be many reasons for poor results. Here we list some of the common problems with advice to help you get the best staining possible along with recommended reading if you want more information:
Direct Staining Flow Cytometry Protocols
This is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens
Indirect Staining Flow Cytometry Protocols
If there is not a directly labeled antibody available, or you wish to amplify your signal you can do indirect staining. This is where you stain a cell with a primary antibody against the antigen of interest and visualize using a labeled secondary antibody which recognizes the primary.
Intracellular Staining Flow Cytometry Protocols
In order to detect antigen not present on the cell surface, cells have to be fixed and permeabilized to disrupt the cell membrane and allow entry of the antibody. Antigens can be then directly or indirectly labelled. Various methods are optimal depending on the antigen and antibody used.
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Direct staining of intracellular antigens: Leucoperm™ Accessory Reagent method
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Direct staining of intracellular CD68: Leucoperm™ Accessory Reagent method
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Direct immunofluorescence staining of intracellular cytokines in blood
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Direct immunofluorescence staining of intracellular antigens: Methanol plus Leucoperm™ Accessory Reagent method
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Direct immunofluorescence staining of intracellular antigens: Digitonin method
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Direct immunofluorescence of intracellular antigens: Paraformaldehyde / Saponin method
Cell Cycle Staining Flow Cytometry Protocols
Measuring DNA content for cell cycle analysis requires fixation and permeabilization of the nuclear membrane. We have protocols for staining using DNA binding dyes with and without antibody staining and a protocol for BrdU staining.
Cell Preparation Flow Cytometry Protocols
Below are protocols for harvesting cells from various sources to obtain healthy cells, essential for optimal staining and analysis.
Product Specific Flow Cytometry Protocols
Specific protocols to use with Bio-Rad antibodies.
Cell Activation Protocols
General activation protocols using pharmacological reagents and antibodies. Ideal to determine immune competence, marker upregulation, cytokine release and proliferation by flow cytometry.
Direct Immunofluorescence Staining of Cells with StarBright Dyes
This method provides a general procedure for use with StarBright Dyes, for staining cells in tubes or a 96-well plate. In some cases, specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the product and batch specific information provided with each vial.
A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications or cell types.
Panel Builder
Create flow cytometry panels with ease using this simple tool...
Spectraviewer
Check the excitation and emission of any fluorophore for instrument and multicolor panel compatibility using this customizable mobile device enabled spectraviewer.
