- Proteins and Peptides
- Lysates and Cell Lines
1. Know your cytometer
The configuration of flow cytometers including light sources (lasers) and optics (mirrors, filters, and detectors) vary and a few, such as the Propel ZE5 Cell Analyzer, can detect up to 30 parameters in thousands to millions of cells. Lasers excite fluorochromes at fixed wavelengths and the emitted fluorescence is captured by optical detectors called photomultiplier tubes (PMTs). Setting PMT voltages correctly is important because a low setting can compromise signal detection. Furthermore, some instruments have interchangeable/swappable filters offering more flexibility when designing flow cytometry experiments.
2. Optimize antibody selections
To prevent complications during your experiment, take time to choose your antibodies and optimize their working conditions. Best practices dictate lowly expressed proteins or those with rare epitopes should be paired with antibodies conjugated to bright fluorophores, and vice versa. Also, test a few different antibody dilutions to determine which concentration has minimal background staining without significant loss of specific signal. Nonspecific binding can arise from saturated antibody concentrations or from interactions between primary antibodies and Fcy receptors on the cell surface.
3. Minimize spectral overlap with compatible fluorochromes
Avoid spectral overlap and minimize compensation issues by carefully selecting fluorochromes for your panel. The Novus Panel Builder enables researchers to easily choose fluorochromes that are compatible with their chosen instrument and visualize the spectra of individual fluorochromes using a customized Spectra Viewer. Ideally, you should select fluorophores with little to no emission spectra overlap, though this becomes more challenging as panel size increases. We also recommend choosing fluorochromes with narrow excitation and emission spectra, as well as taking advantage of the full spectrum of fluorochromes permitted on your instrument. While green or red emitting fluorochromes remain the preferred antibody conjugates, ultraviolet (UV) and near infrared (NIR) fluorochromes are gaining in popularity.
Comparison of fluorochrome combinations with some spillover into adjacent channels to those with little to no overlap. Fluorochrome examples are Alexa Fluor 488, PE, and PerCP-Cy5.5.
4. Incorporate necessary controls
There are several controls you should include in a flow cytometry experiment to validate your results. For instance, viability dyes are important for identifying dead cells which can contribute to nonspecific staining and lead to false positive results. Compared to using side scatter (SSC) and forward scatter (FSC), viability dyes are more effective at gating out, thereby excluding, dead cells from analysis. Examples of viability dyes include Calcein AM, PI, and 7-AAD. Key negative controls such as FMO (fluorescence minus one) controls and isotype controls help to determine the amount and source of fluorescence background.
5. Generate multiple iterations of your multicolor panel
A “perfectly” designed flow cytometry panel may not work as expected when it is carried out. Thus, it’s critical to make multiple iterations of a multicolor panel for testing, especially before using any precious biological samples.