Max FURA-2 Ratio 2.0- 1.6- 1.2- 0.8 0.4- 0.0 РОН HO+ -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 logM Capsaicin Figure 1. Data are mean ± standard deviation (SD); n = 3 replicates. Table 1. Capsaicin concentration (M) Student name Max Fura-2 ratio Diluted supernatant Undiluted supernatant Chris 0.79 Yan 1.40 Ouda 0.38
A group of third-year students, who had been growing chilli peppers at home, decided to bring samples to their Pharmacology practical where they would be measuring capsaicin, to determine which of them had grown the hottest peppers. The three students ground a sample of their peppers in 100 µl of distilled water, centrifuged the solution and then transferred the supernatant into a collection tube. One student used 70 % ethanol from a wash bottle on their bench instead of using distilled water as the extraction fluid. Samples of the collected supernatants were added to a 96 well test plate along with a range of serially diluted capsaicin standards. The students used a FlexStation which automatically added 20 µl aliquots of each supernatant onto separate wells containing 80 µl of cultured cells, which had been transfected with the capsaicin sensitive TRPV1 ion channel and preloaded with the ratiometric calcium indicator dye FURA-2 prior to the class. The FlexStation was programmed to record the maximum FURA-2 ratio after the addition of the sample. The students constructed a capsaicin standard curve in PRISM using the maximum FURA-2 ratios of the capsaicin standards (see Figure 1 below).
The maximum FURA-2 ratios of each of the student's samples are shown in Table 1. Using the standard curve provided, estimate the concentration of capsaicin (M) in each of the diluted and undiluted chilli supernatants.
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