Hoechst 33342 stock solution

A 1 mg/mL (3.14 mM) Nile Red stock solution was prepared by dissolving 1 mg of Nile Red (ThermoFisher, N1142) in 1 mL of DMSO. A 1 mM Vybrant DiD stock solution (ThermoFisher, V22887) and a 20 mM Hoechst 33342 stock solution (ThermoFisher, 62249) were purchased directly. Cells were first stained in 2 μg/mL (6.28 μM) Nile Red in PSW for 20 minutes, then washed in PSW. Cells were then stained in 2.5 μM DiD and 20 μM Hoechst 33342 in PSW for 20 minutes, then washed in 20 μM Hoechst 33342 in PSW. Finally, cells in 20 μM Hoechst 33342 in PSW were loaded into the cage traps and imaged immediately. We found that Hoechst did not stain well under the conditions tested, so we opted to leave it in the solution during imaging to maximize the fluorescence signal.

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.