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Selecting Control Samples for FCS Assays

In order to successfully analyze FCS data, several control measurements should be made to verify you results. The precautions outlined below will ensure that your FCS analysis yields unambiguous results.

• Measure the buffer alone: This will give you information on the background signal from the buffer, or any buffer additive (e.g. BSA or detergent) present in the sample.
• Measure each dye alone in buffer: This will give you useful information on the diffusion time of free dye, and will help you interpret autocorrelation data should there be free dye contaminating your labeled probe.
• Measure each dye in the presence of each unlabeled probe: This will determine if a dye non-specifically interacts with one of the probes being used.
• Measure each labeled probe alone in buffer: This will give you useful information on the diffusion time of labeled probe and will help you decide the effective concentration range that can be used to give optimal fluorescence intensity (counts per second).