1. Preparation of the LysoTracker Red Working Solution

1.1 The first step is to prepare a LysoTracker Red stock solution. The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 1 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 Working Solution Configuration:

Please dilute 1 mM of the storage solution into the working solution using culture medium or a suitable buffer (e.g. PBS). The recommended working solution is between 50 and 100 nanomoles.

Please note: 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: Centrifuge at 1000g for 3-5 minutes at 4°C, then discard the upper layer. The item should be washed twice with PBS, for a duration of five minutes on each occasion.

In order to proceed with the procedure, the cell culture medium must be discarded. Following this, trypsin should be added in order to dissociate the cells and make a single-cell suspension. The cells should then be spun at 1000 g for 3-5 minutes at 4°C. The fluid that has been spun off should then be discarded. The cells should be washed with PBS twice for 5 minutes each time.

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

2.3 The centrifuge should be run at 400g for 3-4 minutes at 4°C. Please dispose of the item immediately.

2.4 The product should be washed twice with PBS, for a duration of five minutes on each occasion.

2.5 The cells should be resuspended in serum-free cell culture medium or PBS, and examined by fluorescence microscopy or flow cytometry.