Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii
Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii
复制标题
模型生物莱茵衣藻脂质含量的荧光测量
DOI：
10.1007/s10811-013-0011-x
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发表时间：
2013-12-01
影响因子：
3.3
通讯作者：
Pan, Junmin
中科院分区：
文献类型：
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作者：
Kou, Zheng;Bei, Shuqing;Pan, Junmin
莱茵衣藻是目前研究最多的微藻之一，有可能作为研究脂类代谢的模型系统。在这种生物体中建立一种快速而简单的中性血脂测量方法是可取的。尼罗红染色对神经脂质的荧光测量已广泛应用于包括微藻在内的各种细胞类型。然而，尼罗红染色用于检测衣藻中性脂质的系统研究尚未见报道。在这里，我们表明尼罗红染色适用于中性脂类的相对和绝对定量，以及可能的大规模筛选脂质积累缺陷的突变株。对影响尼罗红染色的溶剂、细胞浓度、染色时间、尼罗红浓度等因素进行了比较和优化。结果表明，5%二甲基亚砜+1μg/m L−1尼罗红，染色后5~15 m in时间窗可用于1~8 × 10 6个/m L−1细胞内细胞脂质含量的测定。此外，我们开发了一种可能用于大规模筛选具有不同脂质含量的细胞的方案。因此，本文所报道的工作为衣藻作为研究生物柴油生产中脂肪代谢的模式生物提供了及时所需的技术。
The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL−1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8 × 106 cells mL−1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production.