Rhodamine 123 is a fluorescent dye (λex=503 nm, λem=527 nm).

The intensity of R123 fluorescence has a peak at a concentration of 50 μM and decreases to zero at higher concentrations because of self-quenching[1].

Instructions
I. Solution preparation
1. Preparation of mother solution: Dissolve Rhodamine 123 in anhydrous DMSO or ethanol to prepare a 1-5 mM stock solution. ​
Note: Avoid vigorous stirring or shaking during dissolution to prevent fluorescence quenching.
2. Working solution preparation: Dilute the stock solution with appropriate culture medium to the required working concentration, usually 1-2 μM.
II. Operation steps
1. Cell preparation:
1) Culture cells on a slide, the cell density should be 5×10^4 to 5×10^5 cells/mL. ​
2) Wash cells with PBS or Hank’s solution to remove residual culture medium.
2. Staining: Add the working concentration solution to the cells and incubate at 37°C in the dark for 30 minutes to 1 hour. ​
3. Washing:
1) Remove the dye solution and wash the cells with culture medium or PBS to remove unbound dye.
2) If cells need to be fixed, add 10% formalin buffer and incubate for 15-20 minutes, then wash with PBS. ​
4. Observation: Use a fluorescence microscope with a fluorescein filter to observe cells. Rhodamine 123 is excited at a wavelength of about 507 nm and emits green fluorescence at 529 nm. ​
Precautions:
1) Wear gloves during operation to avoid contact between skin or mucous membranes and reagents. ​
2) Avoid light during incubation and storage to prevent fluorescence quenching. ​
3) After staining, fluorescence detection analysis should be performed immediately.