1. Preparation of BODIPY505/515 working solution

1.1 Preparation of BODIPY505/515 stock solution. The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 10 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 Working Solution

Please prepare the BODIPY505/515 Working Solution at a concentration of 1-10 μM by diluting the stock solution with pre-warmed serum-free cell culture medium or PBS. It is recommended that it is used as it is.

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. Suspended Cells

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

2.2 The next step is to add 1 mL of BODIPY505/515 working solution and incubate at room temperature for 5-30 minutes.

2.3 Then, centrifuge at 400 g for 3-4 minutes and discard the upper layer.

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, and observed under the microscope or flow cytometer.

3. 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 5 and 30 minutes.

3.4 The dye working solution should be aspirated, then washed with medium 2-3 times for 5 minutes each time. This process should be observed with a fluorescence microscope or flow cytometer.