Rhodamine 123

1.Preparation of Rhodamine 123 working solution
1.1Preparation of the stock solution
Dissolve 1 mg Rhodamine 123 in 525 μL DMSO to obtain 5 mM of stock solution.
1.2Preparation of Rhodamine 123 working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-20 μM of working solution.
Note: Please adjust the concentration of Rhodamine 123 working solution according to the actual situation.
2.Cell staining
2.1 Suspension cells (6-well plate)
a.Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×106/mL.
b.Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes.
c.Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
d.Wash twice with PBS, 5 minutes each time.
e.Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a.Culture adherent cells on sterile coverslips.
b.Remove the coverslip from the medium and aspirate excess medium.
c.Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 30-60 minutes.
d.Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.
Note: If detection by flow cytometry, cells need to be resuspended before staining.

The cell was seeded into a 6-well plate at a density of 2.5×10⁵ cells/well. After adhered, the cells were then incubated with 10 μM rhodamine 123 or doxorubicin for 2 h at 37°C. After washing three times with ice-cold PBS, cells were analyzed with FCM.