CellTrace Dye Labeling

CFSE and other CellTrace dyes are used to measure relative proliferation rates of cells. These dyes are amine-reactive and cell permeable and thus react with proteins within the cell. As cells divide, the dye is split between the daughter cells and thus diluted through cell division. Lower CFSE signal is indicative of more cell divisions, relative to a reference population.

In our lab, we use CFSE and other CellTrace dyes to measure relative proliferation rates of reprogramming populations. Cells are stained the day after transduction of reprogramming factors (1 day post-infection, 1 dpi). However, this protocol may be applied to any cell population where relative proliferation rates are of interest.

It is possible to stain cells in suspension or on the plate. In suspension uses less dye and may lead to more even staining. The drawback is that with some of the dyes (e.g. CFSE) you start to lose resolution at 4 dpi in NILDDRR conditions.

Note

CFSE (500 µg) is the cheapest by far but NW has observed it may dilute out faster than CTV and CTFR. Specifically, NILDDRR at 4 dpi will be closer to the negative control in CFSE than with other dyes.

CellTrace dyes available in lab

Dye	Attune Channel (excitation/emission)	Stock Concentration	How to Make
CFSE	BL1 (495/519)	10 mM (1000x)	Add 9 or 90 µL of DMSO directly to vial of CFSE. For the Cell Trace Kits, add 9 µL. For the 500 µg vials, add 90 µL of DMSO.
Cell Trace Violet (CTV)	VL1 (405/450)	5 mM (1000x)	Add 20 µL of DMSO directly to vial of CTV Cell Proliferation Kit.
Cell Trace Far Red	RL1 (630/661)	1 mM (1000x)	Add 20 µL of DMSO directly to vial of Cell Trace Far Red Cell Proliferation Kit.

Other materials needed:

• PBS

• DMEM + 10% FBS

• Adherent cells to stain

Important

Always include a negative (unstained) control. When adding + DDRR, you want to know when you’re starting to lose signal.

Note

For MEFs and reprogramming experiments, you may want to work in 24-well plates or larger to have enough cells for flow quantification (>10,000 cells). A typical 96-well of MEFs NIL at 4 dpi will have ~1-10k cells and NIL + DDRR at 4 dpi will have ~10-100k cells. Be careful to remember if you’re doing 6F you will need even more. However, results are normally consistent with 96-wells if careful.

Dilute Cell Trace Dye to working concentration

1. If available, use an already resuspended vial of desired CellTrace dye. If unavailable, or not enough available, resuspend dye in DMSO according to the above table.

   • One entire 24-well plate will require 6 mL of working concentration CellTrace Dye, so you will need 6 µL of stock dye per plate being stained.

2. Dilute stock solution of dye into PBS to make a working solution. Stocks are at 1,000x concentration.

Wash cells and add CellTrace

3. Working in a BSC, aspirate the media and wash with PBS to remove media proteins.

4. Aspirate PBS and replace with working solution of CellTrace. For control cells that do not get stained with CellTrace, incubate in PBS + 1:1,000 DMSO or just PBS.

   • For the CellTrace staining a “half” well volume may be used (e.g. 1 mL per well of 6-well plate, 0.5 mL in a 12-well, etc.)

5. Incubate the cells in CellTrace in the incubator (37°C) for 30 minutes.

Remove CellTrace and wash

6. After incubation, aspirate the CellTrace solution and wash once with FBS containing media.

7. Return cells to incubator and treat cells according to your experiment. For reprogramming, cells are collected at 4 dpi and assayed via flow cytometry.