Preparation of Hoechst 33258 working solution

1.1 Preparation of Stock Solution The stock solution should be prepared using ultrapure water (ddH2O, concentration 1 mg/mL). It should then be aliquoted and refrozen 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 Working Solution

The stock solution should be diluted with pre-warmed serum-free cell culture medium or PBS to a final concentration of 10 μg/mL Hoechst 33258 Working Solution.

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 (Suspended Cells)

2.1 The cells should be collected by centrifugation and then washed twice with PBS for 5 minutes on each occasion.

2.2 The next step is to add 1 mL of Hoechst's Working Solution and incubate for 3-10 minutes at room temperature.

2.3 The centrifuge should be run at 400 g for 3-4 minutes, after which the fluid should be decanted.

2.4 The cells should be washed twice with PBS, with each wash lasting five minutes.

2.5 The cells should be resuspended in 1 mL of serum-free medium or PBS, after which they should be observed using a fluorescence microscope or flow cytometer.

3. Cell staining (adherent cells)

3.1 Culture adherent cells on sterile coverslips.

3.2 The coverslip should be removed from the medium, and the excess medium should be aspirated.

3.3 The next step is to add 100 μL of the dye working solution. This should be shaken gently to ensure complete coverage of the cells, after which the solution should be left to incubate for between three and ten minutes.

3.4 The next step is to aspirate the dye working solution, then wash it with medium 2-3 times for 5 minutes each time. Finally, observe it using a fluorescence microscope or flow cytometer.