基于细胞组学技术提高胶球藻C-169中性脂染色的有效性

Improvement in the Effectiveness of Staining Neutral Lipids in Coccomyxa subellipsoidea C-169 by Cytomics Technologies

本研究基于流式细胞仪、荧光酶标仪、激光共聚焦扫描显微镜等细胞组学分析技术,系统研究了藻液细胞浓度、Nile Red浓度、助溶剂种类和浓度,以及染色温度、染色时间、振荡时间等对检测藻细胞中性脂相对含量（以平均荧光强度表示）的影响,提高了胶球藻C-169中性脂染色的有效性。结果表明,Nile Red荧光染色的最佳藻细胞浓度为106 mL,染料浓度在0.05-0.2μg/mL之间较适宜且不会产生过量的荧光干扰;添加15%-30%（V/V）丙三醇能显著增强染料进入细胞的通透性;25-40℃范围内胶球藻染色后的荧光强度较强但无显著差异（p〉0.05）;染色时间超过10 min及振荡时间超过30 s均未显著提高胶球藻C-169染色后的平均荧光强度。系统优化后的染色方法可从细胞水平上显著提高中性脂荧光检测的精确性,同时为其他微藻采用这一荧光探针快速测定中性脂含量提供了快速有效方法。

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.