Nile red stock solution

1× chemical staining working solutions were prepared with PBS-T solution (1× phosphate buffer saline, pH = 7.4, supplemented with 0.05% Tween-80). Specifically, Nile red stock solution (Sigma, 1 mg/mL in DMSO) was diluted with to a final concentration of 5 μ l/ml; Hoechst 33342 stock solution (Life technologies, 10 mg/mL in distilled water) was diluted to a final concentration of 10 μ g/ml; Syto-17 stock solution (ThermoFisher, 5 mM in DMSO) was diluted to a final concentration of 0.5 μ M; FM4-64 stock solution (ThermoFisher, 1mg/ml in DMSO) was diluted to a final concentration of 10 μ g/ml. 1 mL culture (OD₆₀₀ ≈ 1.0) of wild type Msm cells were spun down and washed with PBS-T solution for two times, then lifted with 1mL PBS-T. Pellets of 200 μ l aliquots were stained with equal volumes of the 1× chemical staining working solutions at room temperature for 10 min. After staining, cells were washed twice with PBS-T, resuspended in 100 μ l PBS-T, and subjected to imaging immediately.