Flow cytometric visualization of cell generations
The CellTrace Violet 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.
This protocol can be used for:
- Detecting cell proliferation using flow cytometry
This protocol should not be used for:
- Fluorescence microscopy or microplate readers
You will need the following for this protocol:
- 10 mL of heparinized peripheral whole blood
- CellTrace Violet Cell Proliferation Kit (Cat. No. C34557)
- OpTmizer T Cell Expansion SFM (Cat. No. A10485-01)
- Penicillin-Streptomycin-Glutamine (100X) (Cat. No. 10378-016)
- PBS (Cat. No. 10010-023)
- GE Ficoll-Paque™ Plus (Fisher Cat. No. 45-001-750)
- Cell counter such as the Countess II FL (Cat. No. AMQAF1000)
- Dynabeads Human T-Activator CD3/CD28 (Cat. No. 11161D)
Protocol
Culture medium preparation
- To 1 L 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
- Complete medium is stable for 4 weeks when stored at 2–8°C in the dark
Mononuclear cell isolation from whole blood
- Dilute 10 mL whole blood in 10 mL PBS and mix well
- Add 15 mL Ficoll-Paque™ Plus to a 50 mL centrifuge tube and gently layer 20 mL diluted whole blood on top
- Centrifuge for 30 minutes at 400 x g
- Carefully remove lymphocyte layer and transfer to a new tube
- Resuspend cells in 25 mL DPBS buffer in a 50 mL conical tube
- Centrifuge for 5 minutes at 300 x g, pour off supernatant, and resuspend in 25 mL DPBS
- Repeat wash step and resuspend in 10 mL DPBS
- Count cells on the Countess Automated Counter or by another method; adjust concentration to 106 cells/mL
Cell staining
- Add 20 µL DMSO to a vial of CellTrace Violet staining solution
- Dilute this stock solution into 20 mL of PBS (warmed to 37°C) for a 5 µM staining solution
- Add 10 mL of cells to a 50 mL centrifuge tube
- Centrifuge cells for 5 minutes at 300 x g and carefully pour off supernatant
- Resuspend cells in 10 mL of CellTrace Violet staining solution
- Incubate cells for 20 minutes in a 37°C water bath
- Add 40 mL OpTmizer T Cell Expansion SFM to the cells to absorb any unbound dye
- Incubate cells for for 5 minutes
- 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
- Distribute aliquots of stained cells into culture plates or flasks
- Stimulate with 50 µL Dynabeads Human T-Activator CD3/CD28 per 1 mL of cells, or other stimulus
- Incubate for desired length of time under growth conditions
- Harvest cells and stain for other markers if appropriate
- Analyze using a flow cytometer with 405 nm excitation and a 450/40 bandpass emission filter
Spectral information and storage
CellTrace Violet | |
---|---|
Excitation/Emission (in nm) | 405/450 |
Standard filter set | Pacific Blue |
Storage conditions | ≤–20°C |
Protocol tips
- Reserve 1 mL of cells for unstained control and 1 mL of cells for a stained, but unstimulated control
- Dynabeads stimulation typically results in T-cell division every 18–20 hr
- Analyze as many cells as possible from each sample
- Use a viability dye and gate on live cells
Human PBMCs stained with CellTrace Violet reagent and stimulated in culture for 7 days. The discrete peaks in this histogram represent successive generations of live cells. Analysis was completed using the Attune Acoustic Focusing Cytometer with 405 nm excitation and a 450/40 nm bandpass emission filter. The unstimulated parent generation is indicated in red.
For Research Use Only. Not for use in diagnostic procedures.