DiOC6(3) iodide (ultra pure)

Prepare DMSO or EtOH stock solutions: The stock solutions should be prepared in DMSO or EtOH at 1-5mM. The unused portion of the stock solution should be stored at -20 °C. Avoid repeated freeze/thaw cycles.

Prepare working solutions: Dilute the stock solutions (from Step 1) into a suitable buffer such as serum-free culture medium, HBSS or PBS to make 1 to 5 µM working solutions. The final concentration of the working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations that are at least over a tenfold range.

DiO and can be used with standard FITC.

Stain the cells in suspension:

1. Suspend cells at a density of 1 x 10⁶/ml in dye working solution (from Step 1.2).
2. Incubate at 37 °C for 2–20 minutes. The optimal incubation time varies depending on the cell type. Start by incubating for 20 minutes and subsequently optimize as necessary to obtain uniform labeling.
3. Centrifuge the labeled suspension tubes at 1000 to 1500 rpm for 5 minutes.
4. Remove the supernatant and gently resuspend the cells in pre-warmed (37 °C) growth medium.
5. Wash two more times as Steps 3 and 4.

Stain adherent cells:

1. Grow adherent cells on sterile glass coverslips.
2. Remove coverslips from growth medium and gently drain off excess medium. Place coverslip in a humidity chamber.
3. Pipet 100 μL of the dye working solution (from Step 1.2) onto the corner of a coverslip and gently agitate until all cells are covered.
4. Incubate the coverslip at 37 °C for 2–20 minutes. The optimal incubation time varies depending on the cell type. Start by incubating for 20 minutes and subsequently optimize as necessary to obtain uniform labeling.
5. Drain off the dye working solution and wash the coverslips two to three times with growth medium. For each wash cycle, cover the cells with pre-warmed growth medium, incubate for 5-10 minutes and then drain off the medium.