DiSC3(5)

1. Preparation of DiSC3(5) Membrane Staining Solution:
1.1 Preparation of DMSO or EtOH Stock Solution: The stock solution should be prepared at a concentration of 1-5 mM in DMSO or EtOH.
Note 1): Unused portions of the stock solution should be stored at -20°C. Avoid repeated freeze-thaw cycles.
1.2 Preparation of Working Solution: Dilute the stock solution in a suitable buffer, such as serum-free culture medium, HBSS, or PBS, to prepare a working solution of 1 to 5 μM.
Note 2): The concentration of the working solution should be determined empirically for different cell types and/or experimental conditions. Testing a range of concentrations, at least tenfold, is recommended.
2. Staining Cells in Suspension:
2.1 Suspend cells at a density of 1 × 10⁶/mL in the dye working solution.
2.2 Incubate at 37°C for 2-20 minutes. Incubation time depends on the cell type. Start with a 20-minute incubation and then optimize as needed to obtain uniform labeling.
2.3 Centrifuge the labeled suspension tube at 1000 to 1500 rpm for 5 minutes.
2.4 Remove the supernatant and gently resuspend the cells in pre-warmed (37°C) growth medium.
2.5 Wash twice following steps 2.3 and 2.4.
3. Staining Adherent Cells:
3.1 Culture adherent cells on sterile glass coverslips.
3.2 Remove the coverslip from the growth medium and gently drain off excess medium. Place the coverslip in a humidified chamber.
3.3 Pipette 100 μL of the dye working solution onto the corner of the coverslip and gently swirl until all cells are covered.
3.4 Incubate the coverslip at 37°C for 2-20 minutes. Incubation time varies depending on the cell type. Start with a 20-minute incubation and then optimize as needed to obtain uniform labeling.
3.5 Drain off the dye working solution and wash the coverslip two to three times with growth medium. For each washing cycle, cover the cells with pre-warmed growth medium, incubate for 5-10 minutes, and then remove the medium.
4. Microscopic examination.
5. Flow cytometry analysis:
DiS-labeled cells can be analyzed using the standard FL3 flow cytometry detection channel.