5(6)-cFDA标记乳酸菌细胞的影响因素

Factors Affecting the Labeling of Lactic Acid Bacterial Cells with 5-(and) 6-Carboxyfluorescein Diacetate

选用常规发酵乳菌种,探讨不同溶剂、不同浓度的羧基荧光素二醋酸酯(5-(6)-Carboxyfluorescein diacetate,5(6)-cFDA)标记乳酸菌细胞的影响因素。结果显示:水、PBS和二甲基亚砜(DMSO)配制的5(6)-cFDA标记初期,细胞标记率分别为94.8%、95.2%和99.77%,放置30d后细胞标记效果分别下降到51.53%、55.6%和96.7%;每毫升菌悬液加60μL的5mg/mL 5(6)-cFDA(相当于终浓度为6.52×10-7mol/L),其荧光强度与5(6)-cFDA浓度成正比,且标记效果最理想。实验还证实了1×106～1×107cells/mL是流式细胞仪检测细胞活性较适合的浓度,且标记率较高;在pH7.0的条件下对细胞活性和发光强度影响最小,其标记率达到98.3%。

The co-cultured cells of Lactobacillus bμlgaricus1.1480 and Streptococcus thermophilus6038 were labeled with different concentrations of 5-（and） 6-carboxyfluorescein diacetate（5（6）-cFDA） in different solvents in order to explore the affecting factors of 5（6）-cFDA labeling.The initial percentages of lactic acid bacterial cells labeled with 5（6）-cFDA in double distilled water,PBS or dimethyl sulfoxide（DMSO） were 94.8%,95.2% and 99.77%,respectively,which were decreased to 51.53%,55.6% and 96.7% after 30 days.The cell suspension at a cell density of 1.0×106 cells/mL was added with different volumes of 5 mg/mL 5（6）-cFDA to desired final concentrations（1.09×10-7,2.17×10-7,4.35×10-7,6.25×10-7,8.7×10-7 and 10.87×10-7 mol/L）.The diluted cell suspension containing 6.52×10-7 mol/L 5（6）-cFDA showed the best labeling efficiency and there was a linear relationship between cell labeling efficiency and the final concentration of 5（6）-cFDA ranging between 1.09×10-7 and 6.25×10-7 mol/L.It was also demonstrated that a cell density between 1×106 and 1×107 cells/mL was suitable for flow cytometric（FCM） determination of living cells,with a high labeling efficiency.The buffer at pH 7 had the smallest effect on cell viability and fluorescence intensity and the resulting labeling efficiency reached 98.3%.