Protocol: Measuring Cell Proliferation Using CytoTrack, a Cell-Permeable Dye

Reagents

• Cell culture medium
• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• CytoTrack Cell Proliferation Assay Kit (1351202, 1351203, 1351205)
• Dimethylsulfoxide (DMSO)
• Required when staining cell surface antigens:
  - Staining Buffer (BUF073)
• Optional: Fixable live/dead dye

Methods

1. Prepare cells appropriately. Refer to protocol FC1 for information on preparation of cells from tissue culture cell lines, FC2 for isolation of peripheral blood mononuclear cells from whole blood, and FC3 for preparation of cells from tissues.
2. Resuspend cells in PBS and adjust to 2 x 10⁶ cells/mL.
3. Prepare a 500x stock solution of CytoTrack Dye by adding 50 µL DMSO to the vial and mixing.
4. Aliquot 500 μL of the cell suspension.
5. Add 1 µL of the prepared CytoTrack Dye stock solution to the cells.
   Note: Add 1 µL DMSO as a vehicle-only negative control.
6. Incubate at room temperature for 15 min while avoiding direct light.
7. Add 3 mL of cell culture medium, centrifuge at 300–400 x g for 5 min, and discard supernatant.
8. Resuspend cells in 500 µL cell culture medium.
9. Incubate cells in a humidified 37^ᵒC, 5% CO₂ incubator for 48–96 hr, as required.
10. Transfer culture to 5 mL tubes and centrifuge at 300–400 x g for 5 min.
11. Remove supernatant and resuspend at 1 x 10⁶ cells/mL.
12. Perform staining of cell surface antigens (protocol FC4), as required along with associated wash steps.
13. Acquire samples on a flow cytometer.

Notes

• CytoTrack Cell Proliferation Assay Kit is available in three formats: 403/454 (1351202), 511/525 (1351203), and 628/643 (1351205). The names refer to the excitation and emission wavelengths. The most appropriate option for your assay and cytometer configuration should be used
• CFDA-SE Cell Proliferation Assay Kit (1351201) can be used as an alternative to CytoTrack Cell Proliferation Assay Kit
• All antibodies should be titrated prior to use to ensure the optimal concentration is used
• Appropriate controls should be included; for example, an unstimulated control is important to include in a stimulation assay
• To allow gating on live cells during analysis, including viability dye staining as part of step 12 is recommended. The most appropriate live/dead dye depends on the fluorophores used and if the cells are to be fixed. A fixable dye, such as VivaFix Cell Viability Assay (1351111), is required for intracellular staining of fixed cells