2.8. 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein (DAF-FM) staining

Briefly, a 5 mM DAF-FM stock solution (Invitrogen) was prepared by dissolving in dimethyl sulfoxide (DMSO), and a 5 μM working solution in PBS was prepared. After removal of the supernatant, the working solution was added, and the VSs were incubated for 1 h at 37 °C in the dark. Then the probe was replaced with PBS, and the VSs were incubated for another 30 min before they were imaged using the microscope with green fluorescence channel (Nikon, eclipse TE2000-s) or quantified at ex/em 485 nm/528 nm using a microplate reader.