The extinction coefficient of the Chl a at 445 nm was calculated using a published extinction coefficient at 663 nm (ε663 nm = 82.04) [30 (link)] using Chl a prepared from various sources, including tobacco leaf, N. oceanica, and P. tricornutum. The absorbance values at 445 nm and 663 nm were measured and a calibration curve was established. This allowed for us to estimate the extinction coefficient of Chl a at 445 nm.
For detecting the concentration of fucoxanthin in P. tricornutum using a spectrophotometry, the cultures were diluted with culture medium, and the absorbance measured at 750 nm (A750 ranges from 0.1 to 0.8). In parallel, a volume of culture was centrifuged and the cells resuspended in an equal volume of ethanol, then the A445 and A663-values were detected after dilution with ethanol (A445 & A663 range from 0.2 to 1). Samples were protected from light exposure as much as possible using foil. The cells were suspended in ethanol and analyzed at A445 and A663 within 5 min. With this data, the concentration of fucoxanthin could be calculated using our formula.
A multi-mode microplate reader (SynergyTM HT, BioTek, Winooski, VT, USA) with 96-well plates was used to study the feasibility of high-throughput analysis following the method described above with the following modifications. The volume of samples in each well was 200 μL. We corrected to a 1 cm path-length using the software provided with the plate reader.
For detecting the concentration of fucoxanthin in P. tricornutum using a spectrophotometry, the cultures were diluted with culture medium, and the absorbance measured at 750 nm (A750 ranges from 0.1 to 0.8). In parallel, a volume of culture was centrifuged and the cells resuspended in an equal volume of ethanol, then the A445 and A663-values were detected after dilution with ethanol (A445 & A663 range from 0.2 to 1). Samples were protected from light exposure as much as possible using foil. The cells were suspended in ethanol and analyzed at A445 and A663 within 5 min. With this data, the concentration of fucoxanthin could be calculated using our formula.
A multi-mode microplate reader (SynergyTM HT, BioTek, Winooski, VT, USA) with 96-well plates was used to study the feasibility of high-throughput analysis following the method described above with the following modifications. The volume of samples in each well was 200 μL. We corrected to a 1 cm path-length using the software provided with the plate reader.
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