Improvement in the Effectiveness of Staining Neutral Lipids in Coccomyxa subellipsoidea C-169 by Cytomics Technologies
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Abstract:
Using cytomics technologies, such as flow cytometry, fluorescence microplate reader, and confocal laser scanning microscope, the effects of algal cell concentration, nile red concentration, types and concentration of assistant solvents, staining temperature and time as well as shaking time on the relative content of neutral lipids in algal cells (expressed as average fluorescence intensity) were investigated. The effectiveness of staining neutral lipids in Coccomyxa subellipsoidea C-169 was improved. The results indicated that the optimal algal cell concentration for nile red fluorescence staining was 106 cells/mL, and the optimal dye concentration ranged from 0.05 to 0.2 μg/mL, which could avoid interference due to excessive fluorescence. The addition of 15~30% (V/V) glycerol could significantly improve the permeability of dye into the cells. Relatively high fluorescence intensity was detected at the staining temperatures ranging from 25 to 40 ℃, but no significant difference was found among them (p>0.05). The average fluorescence intensity of Coccomyxa subellipsoidea C-169 after staining was not significantly improved when the staining time was increased to over 10 min and the shaking time was increased to over 30 s. The systematically optimized staining procedure can significantly improve the accuracy of fluorescence detection of neutral lipids at the cellular level, and provides an effective method for rapid determination of the neutral lipid content in other microalgae using this fluorescence probe.
