CellTrace™ CFSE Cell Proliferation Kit Protocol

The Invitrogen™ CellTrace™ CFSE kit is used to monitor distinct generations of proliferating cells by dye dilution. Live cells are covalently labeled with a very bright, stable dye. Every generation of cells appears as a different peak on a flow cytometry histogram.

Culture medium preparation

1. To 1 L Gibco™ OpTmizer™  T Cell Expansion SFM, add the following:
   • 26 mL of T Cell Expansion Supplement (supplied in Cat. No. A10485-01)
   • 10 mL of Penicillin-Streptomycin-Glutamine
2. Complete medium is stable for 4 weeks when stored at 2–8°C in the dark

Mononuclear cell isolation from whole blood

1. Dilute 10 mL whole blood in 10 mL PBS and mix well
2. Add 15 mL Invitrogen™ Ficoll-Paque™ Plus to a 50 mL centrifuge tube and gently layer 20 mL diluted whole blood on top
3. Centrifuge for 30 minutes at 400 x g
4. Carefully remove lymphocyte layer and transfer to a new tube
5. Resuspend cells in 25 mL DPBS buffer in a 50 mL conical tube
6. Centrifuge for 5 minutes at 300 x g, pour off supernatant, and resuspend in 25 mL DPBS
7. Repeat wash step and resuspend in 10 mL DPBS
8. Count cells on the Invitrogen™ Countess™ Automated Counter or by another method; adjust concentration to 10⁶ cells/mL

Cell staining

1. Add 18 µL DMSO to a vial of CellTrace CFSE staining solution
2. Dilute this stock solution into 20 mL of PBS (warmed to 37°C) for a 5 µM staining solution
3. Add 10 mL of cells to a 50 mL centrifuge tube
4. Centrifuge cells for 5 minutes at 300 x g and carefully pour off supernatant
5. Resuspend cells in 10 mL of CellTrace CFSE staining solution
6. Incubate cells for 20 minutes in a 37°C water bath
7. Add 40 mL OpTmizer T Cell Expansion SFM to the cells to absorb any unbound dye
8. Incubate cells for 5 minutes
9. Centrifuge cells for 5 minutes at 300 x g and resuspend the cell pellet in pre-warmed OpTmizer T Cell Expansion SFM

Stimulation and analysis

1. Distribute aliquots of stained cells into culture plates or flasks
2. Stimulate with 50 µL Invitrogen™ Dynabeads™ Human T-Activator CD3/CD28 per 1 mL of cells, or other stimulus
3. Incubate for desired length of time under growth conditions
4. Harvest cells and stain for other markers if appropriate
5. Analyze using a flow cytometer with 488 nm excitation and emission filters appropriate for fluorescein

Human T lymphocytes stained with the CellTrace™ CFSE Cell Proliferation Kit and stimulated in culture for 5 days. The discrete peaks represent successive generations of live cells. The unstimulated parent generation is indicated in blue. Analysis was completed using an Attune® Acoustic Focusing Cytometer with 488 nm excitation and a 530/30 nm bandpass emission filter.