Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions

We have studied the degree to which fluorescent Ca²⁺ indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca²⁺ indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca²⁺ indicator Fura Red; EGFP can be used with the Ca²⁺ indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca²⁺ signals in cells transfected with fluorescent proteins.