Cell Proliferation Tracer Kit, (Fluorometric, Blue 520)

The following protocol is a general labeling procedure. Because of differences in cell types and variations in culture conditions, optimization of the dye concentration, staining time, and/or staining temperature may be necessary. Higher dye concentrations may be required to track more cell generations, while lower concentrations may be sufficient to track fewer divisions. We recommend using the lowest dye concentration that yields sufficient signal for your assay because cell proliferation dyes can be toxic to cells at high concentrations.

1. Prepare a cell proliferation dye stock solution by dissolving one vial of Cell Proliferation Tracer Dye, Blue 520 in 20 μL of anhydrous DMSO. This brings the stock solution concentration to 5mM. Protect dye stock solutions from light.

Note: Cell Proliferation Tracer dyes are susceptible to hydrolysis. Therefore, the DMSO stock solution should only be prepared on the day of use, and not subjected to freeze/thaw cycles. The dyes should only be added to aqueous buffer immediately before staining. Do not use buffers containing Tris or other free amines.

2. Pellet cells by centrifugation and aspirate the supernatant.

3. Resuspend the cells at 10⁶ cells/mL in pre-warmed (37°C) PBS (or similar buffer) containing 1 μM cell proliferation dye. Protect cells from light for this and all subsequent steps.

Note: Staining can be performed in cell culture medium containing serum, however, this results in 5-10 fold lower fluorescent signal compared to labeling in buffer without serum or other proteins.

4. Incubate the cells for 10-15 minutes at room temperature or 37°C, to allow dye uptake.

5. Add an equal volume of cell culture medium and incubate for 5 minutes at room temperature or 37°C to hydrolyze free dye.

6. Pellet the labeled cells by centrifugation and resuspend in an equal volume of fresh pre-warmed cell culture medium.

7. Incubate the cells for 15-30 minutes at 37°C to allow the dye to react with intracellular proteins.

8. Pellet the labeled cells by centrifugation and resuspend in an equal volume of fresh pre-warmed cell culture medium. Proceed to flow cytometry analysis (step 10). Alternatively, return cells to incubator and culture for the desired period of time to allow cells to divide.

9. Optional: perform formaldehyde fixation, permeabilization, and/or immunostaining.

10. Analyze by flow cytometry in the appropriate channel.

1. Wallace, P.K. et al. (2008) Cytometry A 73, 1019-34.
2. Quah, B.J. and Parish, C.R. (2012) J Immunol Methods 379, 1-14.
3. Tario, J.D. et al. (2011) Methods Mol Biol 699, 119-64.