LysoSensor PDMPO

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare PDMPO staining solution
(1) Dye stock solution: Use DMSO to dissolve PDMPO into a 1 mM stock solution.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C to avoid repeated freezing and thawing.
(2) Working solution preparation: Dilute the stock solution with a suitable sterile buffer (such as serum-free medium or PBS). The recommended working solution concentration is at least 1 µM. Please adjust the concentration of the working fluid according to the actual situation and prepare it now.
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 of working solution preheated at 37°C to resuspend approximately 106 cells, and incubate in the incubator in the dark for 0.5-2h. 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 microscopy 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 100uL 37°C preheated dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.
(4) Incubate in the incubator away from light for 0.5-2h. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(5) After the incubation, discard the dye working solution and wash the coverslips 2 to 3 times with preheated culture solution.
4. Fluorescence detection: PDMPO has pH-dependent dual excitation and dual emission spectrum peaks, producing blue fluorescence (Ex/Em=329nm/440nm) in a neutral environment and yellow fluorescence (Ex/Em=384nm/540nm).
Notes:
①If 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;
②Fluorescent dyes all have quenching problems. Please try to avoid light as much as possible to slow down fluorescence quenching;
③For your safety and health, please wear laboratory clothes and disposable gloves when operating.