Evaluating Intracellular Rhodamine 123 by Flow Cytometry

Intracellular content of rhodamine 123 (Sigma-Aldrich, St. Louis, MO, USA) was evaluated by flow cytometry, as previously reported [36 (link),37 (link)]. The working rhodamine 123 solution was freshly prepared prior to each experiment by dissolving 1 mg in distilled water and then diluting in a complete culture medium to the final concentration of 5 µM. Briefly, the treated and control cells were removed from the culture flask using TrypleTM Express solution (GIBCO, Waltham, MA, USA), spun down, and pelleted. The cells were then resuspended in 1 mL of rhodamine 123 solution in plastic Falcon tubes. The samples were incubated for 1 h at 37 °C in a CO₂-incubator. Following the incubation time, the cells were washed once with ice-cold HBSS (4 °C) (Hank’s Balanced Salt Solution, Lonza, Basel, Switzerland) and then resuspended in 0.5 mL of ice-cold HBSS. The samples were immediately analyzed with a CyFlow^® SPACE flow cytometer (Sysmex, Kobe, Prefektura Hyōgo, Japan) using 488 nm (50 mW) laser excitation and a 536/40 (BP) filter for rhodamine fluorescence detection. The results were analyzed using FCS express 4 flow software (De Novo Software, Glendale, CA, USA). Results were expressed as a mean fluorescence intensity (MIF) and normalized to the control (E0) according to previous study [37 (link)]. Experiments were repeated a minimum of three times.