5 6 carboxydiacetate fluorescein succinimidyl ester cfda

Cell membrane damage was analyzed using the method described by Collado et al. (Collado et al., 2017 (link)). The bacterial cell suspension was double dyed using propidium iodide (PI) (final concentration 10 mM) (Sangon, China) and 5(6)-carboxydiacetate fluorescein succinimidyl ester (CFDA) (final concentration 10 μM) (Beijing Solarbio Science & Technology Co. Ltd., China). Bacterial cell forward scatter, lateral astigmatism, and fluorescent channels FL1 (green) and FL2 (red) were determined using a flow cytometer BD FACSVerse™ (Becton, Dickinson and Company, USA). 1,000 cells were detected in each sample.
The cell membrane hydrophobicity and fluidity assays were performed as described by Pelletier et al. (Pelletier et al., 1997 (link)), and Voss and Montville (Voss and Montville, 2014 (link)), respectively.

Bacterial cell surface hydrophobicity and membrane fluidity were measured according to the methods by Krausova et al. [33 (link)] and Kuhry et al. [34 (link)], respectively. In the former method, 1 mL of 98% cetane (Sangon, Shanghai, China) was added into 1 mL of bacterial cell suspension (OD_{600 nm} values of 0.55 to 0.60) and rotated for 1 min and then stood at room temperature for 30 min. The absorbance of the aqueous phase was measured at OD_{600 nm} using BioTek Synergy 2 (BioTek, Burlington, VT, USA). To measure the membrane fluidity, a 200 μL/well of bacterial suspension was mixed with 2 μL of 10 mM 1,6-diphenyl-1,3,5-hexatriene (DPH) (Sangon, China), and the change of fluorescence intensity of each well was measured at excitation light wavelength of 362 nm and emission light wavelength of 427 nm using BioTek Synergy 2 (BioTek, Burlington, VT, USA).
Cell membrane damage was examined according to the method described previously [32 (link)]. Briefly, the bacterial cell suspension was double-dyed using propidium iodide (PI, 10 mM final concentration) (Sangon, China), and 5(6)-carboxydiacetate fluorescein succinimidyl ester (CFDA, 10 mM final concentration) (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China), and determined using Flow Cytometer BD FACSVerse™ (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) [32 (link)].