Tips and Tricks for Flow Cytometry and FACs

Running flow cytometry, especially FACS, or fluorescence-activated cell sorting, can be tricky for new scientists. The following tips and tricks will help save your team time and effort when performing flow cytometry and FACS, while also improving confidence throughout the experiment and analysis processes.

1. Familiarize Yourself with Your Cytometer(s)

   The first step in preparing for your cytometry experiment is determining 1) whether you have access to a conventional flow cytometer or spectral flow cytometer, and 2) which instrument is most appropriate for your project. Spectral flow cytometers are ideal for high-throughput characterizations of large, multicolor panels but can be more costly.

   Next, before ordering any fluorophores, find out what lasers are available on your instrument of choice. More lasers will be required for larger panels, or panels requiring minimal fluorophore spillover. Finally, consider what biosafety requirements are in place for your cytometer of interest. If your samples cannot be fixed prior to analysis, the risk of aerosolization during sample aspiration will determine what safety measures will be necessary.

2. Designing Your Panel Efficiently

   If you are designing a multicolor panel for your flow cytometry or cell sort, single-color controls are also a must. In these cases, compensation can be completed using either antibody binding beads or single-color stained controls. If necessary, fluorescence minus one (FMO) controls can also be used, particularly for your first time testing a new marker. Best practice involves choosing brighter fluorophores (i.e. PE) for lower expression cell surface markers and vice versa. Tandem dyes, however, can cause signal spillover of the donor fluorophore into other channels, so make sure to map the fluorescence of your entire panel before beginning.

3. Complete a Pilot Study

   Using properly titrated staining antibody concentrations is essential for successful multi-color flow panels. For cell sorting, given stricter gating practice, a pilot study will ensure sufficient cell numbers can be obtained from your larger experiment.

4. Consider Signal Degradation

   Some cell sorting projects require culturing of cells following sorting. However, depending on the target cell type(s), fluorophores may remain bound in culture. This can 1) effect detection of those markers in future experiments, 2) interfere with cell signaling because of antibody binding, 3) block cell markers or other cell receptors from ligand binding, and/or 4) the fluorophores can degrade or decouple and interfere with other fluorophores. For these reasons, cell sorting panels should be minimized to include only necessary markers.

5. Enrich Rare Cell Types Prior to Analyzing

   Whether you are running standard flow cytometry or cell sorting by FACS, rare cell types can lead to additional reagent waste and unnecessary time at the instrument. One way to achieve a higher frequency of your target population is to use magnetic bead cell separation in order to negatively select for your subset of interest. By enriching your samples prior to analyzing, you will improve the likelihood of catching rare cells in reliable numbers for your project(s).

If you have any questions or concerns when designing your flow cytometry or FACS experiments, don’t hesitate to contact the Biorbyt team for advice on any stage of the process.