1. Preparation of DAPI dihydrochloride working solution

1.1 Preparation of reserve solution The stock solution should be prepared using ultrapure water (ddH20, concentration 1 mg/mL. 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 DAPI dihydrochloride Working Solution

The stock solution should be diluted with serum-free cell culture medium or PBS in order to obtain a working solution of 1-10 μg/mL.

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 Protocol

2.1 Cell Suspension (96-well plate)

2.1.1 Centrifuge at 1000 g for 3-5 minutes at 4°C. Then, discard the upper layer and wash twice with PBS for 5 minutes each time.

2.1.2. Please add 1 mL of working solution and incubate at room temperature for 3-10 minutes.

2.1.3. Centrifuge at 400 g for 3-4 minutes at 4°C. Please dispose of the item immediately.

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

2.1.5. The cells should then be resuspended in serum-free cell culture medium or PBS. The observation of this phenomenon can be conducted using a fluorescence microscope or a flow cytometer.

2.2 Adherent cells

2.2.1. The cells which adhere to the culture should be placed 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 incubate for 3-10 minutes at room temperature.

2.2.4. The cells should be washed twice with medium, for a duration of five minutes on each occasion. The observation of this process should be conducted using a fluorescence microscope or a flow cytometer.