Laurdan  别名：6-十二酰基-N,N-二甲基-2-萘胺

Laurdan（6-十二酰基-N,N-二甲基-2-萘胺）是一种膜透性荧光探针，对与之结合的细胞膜磷脂相具有光谱敏感性，可用于磷脂相的鉴定，Laurdan的吸收波长为340-380nm，发射波长为440-490nm。Laurdan具有膜嵌入性，它的疏水尾部可以插入细胞膜的磷脂双层中，而极性头部留在膜表面附近，能够探测膜内部的极性变化和水分子存在情况。Laurdan具有相态敏感性，在液晶相中的荧光发射会显示较大的红移，而在凝胶相中则会显示较小的红移，能够检测膜的相态和流动性。Laurdan可测量细胞膜的广义极化（GP），高GP通常与膜的低流动性、低极性或高胆固醇含量相关，而低GP则相反。

The following experimental protocol only provides a reference for a Laurdan generalized polarization (GP) measurement experiment of cell membrane. Please modify the working concentration and staining time according to experimental needs or refer to the literature. (from the literature, for reference only)

1. Solution preparation

(1) Storage solution: Dissolve Laurdan in DMSO or chloroform to a final concentration of 5 mM.
Note: After unused storage solution is aliquoted, store it at -20℃ in the dark to avoid repeated freezing and thawing.

(2) Working solution: Dilute the storage solution with experimental buffer (e.g. cell culture medium) to the required working concentration, usually in the range of 1-10 µM.
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.

2. Steps for measuring the generalized polarization (GP) of Bacillus subtilis cell membrane by Laurdan

(1) Cell treatment: Cultivate cells to the middle of the logarithmic growth phase, wash them four times in PBS containing 2% glucose and 1% DMF, resuspend them in the same buffer, and adjust the OD600 to 0.3.

(2) Liposome preparation: Bacillus subtilis polar lipid extract was prepared by detergent dialysis, and the prepared liposome solution was squeezed through a 0.4μm filter membrane 20 times. The 10mg/ml liposome stock solution was diluted to a concentration of 1 mg/ml with Tris buffer (5mM, pH 7.4).

(3) Liposome labeling: Add Laurdan solution to the cell suspension to a final concentration of 10 μM and incubate in the dark for 30 minutes.

(4) Fluorescence measurement: Laurdan fluorescence measurement was performed using a BioTek Synergy MX plate reader. The excitation wavelength was 350 nm, the emission wavelengths were 460 nm and 500 nm, and readings were taken every 2 minutes.

(5) Laurdan fluorescence polarization calculation: Laurdan generalized polarization (GP) value was calculated using the following formula: (I460-I500)/(I460+I500).

References: https://pubmed.ncbi.nlm.nih.gov/29451901/