Practical advice to set up and troubleshoot your flow cytometry experiment for immune monitoring.
The Fluorescence Signal is Too Low
First, choose the detection fluorochrome based on expected expression density. Highly expressed molecules can be detected with less bright fluorochromes, while brighter fluorochromes should be reserved for molecules of low or uncertain expression levels. Second, titrate the chosen fluorochrome to establish the optimal staining conditions and incubation time.
Third, consider using reagents with multiple fluorochromes, like Dextramer® reagents. The higher fluorochrome density increases the signal of positive cells that may be attenuated due to low avidity. Fourth, carry out a dual staining in situations where your target cell is of low abundance and generates low brightness when interacting with your chosen fluorochrome. Using two differently labeled reagents with the same specificity allows visualizing the positive cell population in both signals, appearing on the diagonal in a flow.
Further Help with Troubleshooting Flow Cytometry
Tips and Tricks
Find a collection of various Tips and Tricks to optimize your experiment.
Contact our technical experts for further assistance with troubleshooting.
MHC Dextramer® Reagents
Sensitively quantify target immune cells by flow cytometry with a strong signal-to-noise ratio.