Tips For Flow Cytometry Panel Design
Multicolor flow cytometry is a very powerful tool but panel design is one of its biggest challenges and requires great attention.
5 Tips for flow cytometry panel design
The 5 tips below for your flow cytometry panel design will help optimizing your flow cytometry panel and therefore improving the quality of your data.
1) Know your flow cytometer instrumentation
First things first: You should be aware of the capabilities of the flow cytometer used in your platform because the available lasers and optical filters will dictate the maximum panel size and the fluorochromes or dyes that could be used.
2) Choose the read-outs: Which cell markers are you interested in detecting and quantifying?
Although this tip is not directly related to the technical considerations of panel design, we think it is still relevant especially if you intend to run the same panel multiple times.
In our experience, we have seen many flow cytometrists investing time and effort in setting up and validating a panel and then deciding to go back and changing it after discussing with their Principal Investigator or fellow researchers or after reading an article they hadn’t known about.
This is not to say that a validated panel should be fixed in stone and not evolve based on findings throughout the project, but we think it is key to thoroughly investigate the literature, discuss and consult with fellow researchers in order to ask the relevant questions to be addressed prior to determining the list of cell markers and read-outs in the panel.
3) Select fluorochromes
Determine the most appropriate combination of fluorochromes such that the spectral overlap is minimized. The larger the panel is, the harder this becomes.
Fluorochrome combination choices can be optimized based on the fluorochromes’ normalized emission spectra published by vendors or based on experimentally-generated spillover spread matrices generated by flow core managers. Both options are possible on EasyPanel.
Many excellent online panel design tools exist but EasyPanel is special in that it ranks all possible panels and ultimately suggests the concrete specific ones that minimize spectral overlap between fluorochromes.
Including a cell viability dye is highly recommended in order to eliminate dead cells from the analysis. This is important because dead cells tend to be autofluorescent and could therefore be false positives for multiple read-outs.
If your protocol involves fixing and/or permeabilizing cells, then you should ideally avoid employing fluorochromes that are sensitive to these types of manipulations such as certain tandem dyes.
4) Match antigens to fluorochromes
Now that your fluorochromes combination minimizing spillover or spread is set and optimized, you have to judiciously match them to antigens such that:
-Bright fluorochromes are associated to antigens that typically exhibit low expression on the cell populations of interest, and vice versa.
-Coexpressed antigens (i.e., antigens expressed on the same cell population) are matched to fluorochromes with minimal spectral overlap. Inversely, you can match mutually exclusive antigens to fluorochromes that feature the highest spectral overlap within the panel.
-Check commercial availability of the fluorochrome-conjugated antibodies to purchase readily-available products and avoid the need for in-house conjugation.
5) Experimental Setup
5-1) Prepare your cell sample correctly
-Assess cell viability before performing the experiment
-Keep cells at 4C during staining
-Reduce the formation of cell aggregates by avoiding high cell concentrations and potentially adding DNAse I and EDTA
5-2) Reduce non-specific binding of antibodies by:
-Adding BSA or unconjugated antibodies or FcR blocking reagents in the staining buffer.
-Titrating antibodies (i.e., determining the optimal antibody concentration during staining) to maximize the signal to noise ratio
5-3) Control for potential non-specific binding of antibodies by including isotype controls
An isotype control is the same antibody subclass conjugated to the same fluorochrome (as the test antibody) but against an irrelevant antigen. It is used at the same concentration in a sample control tube.
EasyPanel is an Automated and Intelligent tool for flow cytometry panel design (traditional and spectral cytometers). It is licensed by leading biotech, pharma and academic flow core facilities to design optimized panels of up to 48 colors in seconds or minutes.
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