With the kinetics of WinterGreen-WAY uncaging defined, we sought to test WinterRed-NDMC uncaging under cell culture conditions. As an initial test of WinterRed-NDMC antagonistic efficacy, we designed a competition assay to test for spatially controlled NDMC binding of 5HT2C against WAY-161503. Media was loaded with 10 µM WinterRed-NDMC, 5 µM WAY-161503 was injected into the media, then the entire field of view was irradiated with 639 nm light to uncage NDMC. The calcium flux duration was compared to that of a sample containing no WinterRed-NDMC. A statistically significant decrease in both calcium flux duration and amplitude of calcium signaling was observed (Figure ). As a note, 5 µM WAY-161503 was the upper boundary of successful competition for this assay. Concentrations higher than 5 µM WAY-161503 did not produce a statistically significant change in calcium flux duration or fluorescence amplitude. From this we concluded that the photolysis reaction of WinterRed-NDMC is active in cell culture media and demonstrated spatial modulation of calcium signaling within a large population of 5HT2C expressing cells. Future studies will address the kinetics of WinterRed-NDMC uncaging in cell culture media, though from our initial investigation, we can conclude that 5 seconds at 639 nm is sufficient to outcompete up to 5 µM WAY-161503.