Lyso-Tracker Red (Synonyms: LysoTracker Red DND-99)

This plan only provides a guide, please modify it to meet your specific needs.

1. Prepare Lyso-Tracker Red staining solution

(1) Take out the frozen dyeing solution, return it to room temperature, and concentrate the dyeing solution at the bottom of the tube by instant centrifugation.

(2) Preparation of working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium or PBS) to prepare a working solution with a concentration of 50-75nM.

Notes:

① Please adjust the concentration of the working fluid according to the actual situation and prepare it as needed;

② In order to reduce false positives caused by excessively high background or overload, the concentration of dyes should be as low as possible;

③ If cells are incubated in a buffer free of dye after staining, attenuation of fluorescence signals and vacuolization of cells will be observed.

2. Cell suspension staining

(1) Suspension cells: Centrifuge the suspended cells at 4°C and 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS for 5 minutes each time.

(2) Adherent cells: Wash twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(3) Use 0.5-1mL working solution to resuspend approximately 10⁶ cells, and incubate at room temperature in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.

(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS to wash 2-3 times, 5 minutes each time.

(5) Resuspend cells in pre-warmed serum-free cell culture medium or PBS and observe by fluorescence microscope or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, and place the coverslip in a humid environment.

(3) Add 100μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.

(4) Incubate at room temperature in the dark for 5-30 minutes. The optimal culture time for different cells is different.

(5) After the incubation, discard the dye working solution and wash the coverslip 2 to 3 times with pre-warmed culture solution.

4. Fluorescence detection: The maximum absorption/emission light of Lyso-Tracker Red is 577/590nm.

Notes:

①    A kinetic study on the internalization of Lyso-Tracker revealed that the rate of uptake of this probe by living cells can be completed within a few seconds. However, the probe exhibits a lysosomal "alkalization effect", and prolonged incubation can lead to an increase in lysosomal pH. Therefore, incubating the cell at 37℃ for 1-5 minutes is a useful pH indicator;

②    If the dyeing is not sufficient, it is recommended to increase the dye concentration or extend the dyeing time to allow the dye to aggregate on lysosomes;

③    Fluorescent dyes all have quenching problems. Please try to avoid light as much as possible to slow down fluorescence quenching;

For your safety and health, please wear laboratory clothes and disposable gloves when operating.