Getting started in flow cytometry may seem overwhelming, so we’ve broken the flow cytometry experiment down into seven basic steps.
1. Select your Cytometer
2. Design your panel
3. Optimizing your staining protocol
4. Select your controls
5. Prepare your sample
6. Run your experiment
7. Analyze your data
1. Select your cytometer and understand its configuration.
If you are fortunate enough to work with a core facility that offers multiple flow cytometer choices, then you may need to consult your core manager about which option is best suited for your planned experiment. Once you have chosen a cytometer, it is important to understand the capabilities of your instrument and how its configuration will affect your experiment design. Flow cytometer configurations typically include light sources (lasers) and optics (filters and detectors). The quantity and arrangement of these parts will ultimately determine the number of parameters that can be measured in an experiment. FluoroFinder works directly with your flow core manager to pre-load all your core’s instrument configurations into an intuitive panel design platform. Simply start a new panel and find your core facility and cytometer in the dropdown lists, then click on the purple “View Configurations” button in the upper right corner. Still unsure about which cytometer is right for you? Check out our Flow Cytometer List for details about commercially available cytometer platforms.
2. Design your multicolor experiment panel.
Your panel should incorporate antibodies for each marker of interest and fluorochromes that fit within the configurations of your cytometer. Brighter fluorophores are best suited for cells with the lower antigen expression, whereas dimmer fluorophores should be used with more highly expressed antigens. Additional attention should be paid to reducing “spillover” or fluorescence signal detection across channels. Our database of over 500,000 products and 550 fluorochromes from 60 top suppliers enables researchers to design, save and optimize multicolor flow cytometry panels. Furthermore, integrated machine specific Spectra Viewers with spillover tips guide users through the complexities of panel design.
3. Optimize your staining protocol
It is important to properly titrate all antibodies to ensure that the amount being used is appropriate for each experiment. Be aware that the optimal working concentrations of an individual antibody may be affected by other antibodies in the panel. Also, be sure to prevent non-specific binding of antibodies by incubating samples with appropriate blocking serums. Some intra-cellular antigens may be difficult to stain. Depending on the markers being examined, fixative and permeabilization solutions may be necessary. However, be aware that fixation will kill your cells!
4. Select your controls
Using proper controls is fundamental for any good flow cytometry experiment design. Depending on your experiment, viability dyes, FMO, beads and isotype controls may all be necessary. Fluorescence Minus One staining should be performed to assess the amount of spillover into each channel. This step is especially important when analyzing dim or non-discrete populations. For more information on flow cytometry controls, see our article on Common Mistakes in Panel Design. Also, consider using a dump channel to combine your viability dyes and other controls.
5. Prepare your sample
Poorly prepared samples will lead to false measurements and inaccurate results. Cells must be properly suspended to remove any clumps or “stickiness” and then filtered to remove any dead cell debris that may cause autofluorescence. Of course, it is also necessary to treat cells gently to keep them alive for the experiment. For more tips on sample preparation, check out our Essential Pre-Sort Checklist.
6. Run your experiment
Booking time on flow cytometers can be expensive, so be sure to arrive at your flow core ready to run your samples efficiently. Also, when booking time to use a cytometer, always remember to allocate time for pre-experiment setup and post-experiment cleanup. Maximize your efficiency at the flow core time by following these tips for Getting More Out of Your Flow Core.
7. Analyze your data
Flow cytometry data is analyzed on software using advanced algorithms. There is a wide variety of both open source and commercially available software options that offer specialized tools for data manipulation, visualization, and export. Many of these analysis software tools can even perform automated compensation to account for fluorophore spillover. For a comprehensive list of software options, check out our Flow Cytometry Analysis Software List.
