Before you start
- Titrate the dye: too much is toxic, too little blurs division peaks.
- Keep an unlabelled sample and a labelled, unstimulated sample (day-0 peak).
Reagents and materials
Totals are for 6 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| PBS, protein-free, 37 °C | 1 mL | 1 mL |
| CFSE or CellTrace dye (final 1–5 µM)for 1 mL labelling at 1 µM | 1 nmol | 1 nmol |
| Complete medium with ≥10% FBS (quench and washes) | 10 mL | 10 mL |
Buffers and recipes
Complete RPMI (cRPMI) per 500 mL
| RPMI 1640 | 440 mL |
| Heat-inactivated FBS (10%) | 50 mL |
| L-glutamine, 200 mM (2 mM) | 5 mL |
| Penicillin–streptomycin, 100× (1×) | 5 mL |
| 2-Mercaptoethanol, 55 mM (50 µM) | 0.45 mL |
Optional: 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids. Use within ~4 weeks.
Equipment
- 37 °C water bath or incubator
- Flow cytometer with the right laser (488 nm for CFSE, 405 nm for CellTrace Violet)
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Wash cells
Wash cells twice in protein-free PBS to remove serum.
Label
Resuspend at 1–10 × 10⁶ cells/mL in warm PBS, add dye to 1–5 µM, mix immediately and incubate 10–20 min at 37 °C protected from light.
Quench
Add 5 volumes of cold complete medium (with FBS) and incubate 5 min.
Wash
Wash twice in complete medium to remove unbound dye.
Culture
Plate with your stimulus for 3–5 days.
Acquire
Stain surface markers if needed and acquire. Each division halves the dye intensity.
Critical points
- Add dye quickly and mix well for a tight starting peak.
- Use the unstimulated labelled sample to set the undivided gate.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Broad starting peak | Mix immediately on adding dye; label at a single uniform density. |
| High death | Lower dye concentration and labelling time. |
Safety
Standard laboratory care.
Further reading
- Quah BJC, Parish CR. The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation. J Vis Exp (2010).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.