1. Preparation of Rhodamine 123 working solution

1.1 Preparation of Rhodamine 123 Reserve Solution. The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 5 mM. The stock solution should then be applied to the unused solution by means of aliquoting and refreezing to a temperature of at least -20°C. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)

1.2 Preparation of Rhodamine 123 Working Solution

The stock solution should be diluted with serum-free cell culture medium or PBS in order to obtain a working solution with a concentration of 1-20 μM.

Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.

2. Cell Staining

2.1 Suspended cells

2.1.1. Centrifuge at 4°C for 3-5 minutes at 1000g. Following this, the upper layer should be discarded. The sample should then be washed with PBS twice, for 5 minutes on each occasion. The cell density is 1×10^6/mL.

2.1.2. The working solution should be added to the relevant sample and left to incubate at room temperature for between 5 and 30 minutes.

2.1.3. Centrifuge the sample at 400g at 4°C for 3-4 minutes, then discard the fluid.

2.1.4. It is recommended that the item be washed twice with PBS for five minutes on each occasion.

2.1.5. Cells should be resuspended in serum-free cell culture medium or phosphate-buffered saline (PBS). They should then be observed using a fluorescence microscope or flow cytometry.

2.2 Adherent cells

2.2.1. The cultivation of adherent cells should be carried out on sterile coverslips.

2.2.2. The coverslip should then be removed from the medium and the excess medium aspirated.

2.2.3. Next, add 100 μL of working solution, shake gently to ensure full coverage of the cells, and then incubate for 30-60 minutes at room temperature.

2.2.4. Please ensure that the culture medium is washed twice, for a duration of five minutes on each occasion. The observation of this sample should be conducted using a fluorescence microscope or a flow cytometer.

Please note that if flow cytometry is to be used, cells must be resuspended before staining.