Flow Cytometry Basics
Flow Cytometry Protocols
Novus Panel Builder
Novus Panel Builder Video Tutorial
Multicolor Flow Cytometry Experiments
Flow cytometry is a powerful research technique that facilitates the measurement of multiple parameters in particles such as individual cells. The detection of specific cellular markers involves the use of fluorochrome conjugated antibodies, fluorescent tags and dyes along with a flow cytometer’s optical system to generate and collect the relevant fluorescence spectra (excitation and emission spectra). While the development of high performance cytometers has enabled more thorough characterization and phenotyping of diverse cell populations, it has also made the design of multicolor experiments more complex.
How to Build a Multicolor Panel
When designing a flow cytometry panel, it is important to know the instrument configuration and how to optimally combine multiple fluorochromes to minimize spectral overlap. Panel design has traditionally been a time-consuming process involving extensive research of available reagents, evaluation of proper fluorochrome/antibody pairings, and verification of panel/cytometer compatibility. The Novus Panel Builder Tool automates this process, leading to shortened research timelines and fewer errors. Find the right products for your experiment by following the 3 easy steps below.
Design a Panel Now
1. Select your Institution and Instrument
Institutions along with their respective flow core facilities/labs and instruments are preset in the system. If your institution and/or instrument is not available, click the add your institution, core, or lab link for further instruction.
2. Select Markers for your Experiment
Add your marker (Antibodies, Viability Dyes, Fluorescent Proteins, etc.) and select the target species. Refining your results by host species, isotype, clone and antigen density is optional. If you want to exclude dead cells or cells positive for specific markers, click the add a dump channel button. If you want to verify that the lasers, detectors, and filters are appropriate for your experiment, click the machine configuration button for a visual layout of the configuration.
3. Select the Appropriate Products that Fit your Instrument
mouse over to view a list of products
View products for your markers within the confines of your instrument configuration (lasers and filters) as a digital spreadsheet. The brightness of the fluorochrome is indicated by the number of colored boxes present (1-5) and the number of available Novus reagents is found in parentheses. When you select a fluorochrome, a popup appears displaying a list of relevant products. If you can’t find a product, please contact technical support. For a visual of all the fluorochrome excitation and emission spectra in your panel, click the show Spectra Viewer button.
Panel Summary: Save a Copy of your Panel and Place your Order
Click the add to cart button to immediately purchase your Novus reagents. To save a copy of your panel for your records or to have your panel reviewed, you can also print, export or email your panel.
Design a Panel Now
Tips for Multicolor Panel Design
- Utilize the full spectrum of fluorochromes permitted on your instrument. By selecting fluorochromes that span across the spectral range of the optical system, fluorescence spillover and compensation issues will be minimized. For example, consider using an antibody conjugated to Alexa 405 (Ex max = 401 nm/Em max = 421 nm) along with an antibody conjugated to DyLight 755 (Ex max = 754 nm/Em max = 776 nm).
- Match fluorochrome brightness with antigen density. In general, highly expressed antigens should be paired with dim fluorochromes whereas the brightest fluorochromes should be used for detection of lowly expressed antigens. If testing an antigen with an unknown concentration in a biological sample, start with a bright fluorochrome.
- Validate your assay with the proper controls. Experiments should include a few different controls.
- Unstained cells are used to access cellular autofluorescence, a factor contributing to false positive staining.
- Viability dyes are key for identifying and excluding dead cells from data analysis. Dead cells impact staining by nonspecifically binding antibodies and showing increased autofluorescence.
- Isotype controls and FMOs (fluorescence minus one) are important for measuring background signal. FMOs are samples that are stained for all markers in an experiment except for one and experiments should include each variation to properly set your gates.
Additional Flow Cytometry Support