What is CFSE labeling?

Labeling cells with carboxyfluorescein succinimidyl ester (CFSE) is an elegant technique for measuring cell proliferation. Cell surface proteins are stably labeled and, as cells divide, the fluorescent label is equally divided between daughter cells. This allows a count of the number of rounds of division.

How does CFSE staining work?

Unlike stains that label the lipid membrane of cells, CellTrace™ CFSE dye easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye is designed to provide a consistent signal, even after several days in a cell culture environment.

What is CFSE in immunology?

Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent cell staining dye. CFSE is cell permeable and covalently couples, via its succinimidyl group, to intracellular molecules, notably, to intracellular lysine residues and other amine sources.

What is CFSE flow cytometry?

CFSE is a versatile tool for the fluorescent intracellular labeling of live cells. Labeled cells can be assayed using flow cytometry and fluorescent microscopy. The dye is long lasting and well retained within labeled cells. The result is live cells with an intracellular fluorescent label.

Is CFSE toxic to cells?

CFSE can be toxic to cells and non-optimal CFSE labeling conditions can thus hamper proliferation of cells and obscure interpretation of results. While CFSE is commonly used to assess lymphocyte proliferation, CFSE can be toxic and impair cell division.

What Colour is CFSE?

CFSE (Carboxyfluorescein succinimidyl ester) is a membrane-permeable fluorescent dye. As the dye diffuses into the cell, its succinimidyl group covalently binds to intracellular lysine residues and other free primary amines, producing green fluorescence.

Can CFSE be fixed?

CFSE readily crosses intact cell membranes. Cells labeled with CFSE may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers, such as the Foxp3 Transcription Factor Staining Buffer Set (cat.

Can CFSE readily enter cells?

In our hands, we’ve found that lipophilic dyes such as PKH26 and PKH67 are particularly prone to transferring to adjacent cells in culture conditions; this is because they integrate into the cell membrane and cell membrane exchange occurs quite readily between cells.

Does CFSE stain dead cells?

Thus live cells label with CalcienAM, CFSE, etc. while dead cells do not. It is important to note that unlike cell cycle protocols, this protocol requires that all buffers do not contain detergent or premeabilizing agents (so the PI should be solubilized in PBS/water or the PI will stain all cells.

How long does CFSE last?

CFSE can last several days depending on the level of T cell stimulation and your initial labeling of the T cells. The dye can usually measure 6-7 divisions.

Can flow cytometry detect dead cells?

Loss of membrane integrity is a definitive indicator of cell death in flow cytometric assays. Cells that exclude a dead cell dye are considered viable, while cells with a compromised membrane allow the dye inside into cell to stain an internal component, thus identifying the cell as dead.

How is CFSE used in the labeling of cells?

CFSE is a versatile tool for the fluorescent intracellular labeling of live cells. Labeled cells can be assayed using flow cytometry and fluorescent microscopy. The dye is long lasting and well retained within labeled cells. The provided CFSE is sufficient for ~1000 assays. CFSE is a cell permeant, non-fluorescent pro-dye.

Is the CFSE protocol safe for human cells?

As you can see, the protocol is relatively simple. CFSE is toxic to cells. I would use couple of concentration, incubate at 37 for about 5 min, and inactivate CFSE by FBS. Also, label cells with some general T cell marker or live stain before flow. You can use it to clean up the signal. Actually, CFSE is not the best out succinimidyl esters.

What’s the difference between CFSE and flow cytometer?

CFSE is a less direct but very simple assay – label cells, wash away dye, examine dye dilution over time (as cells divide the dye is diluted – less dye per cell = dividing cells). These are longer timepoint assays, typically days post labeling. Needs flow cytometer for analysis.

Can a CFSE labeled cell be detected with FITC?

CFSE labeled cells can be detected with any instrument or filter set compatible with FITC detection: Excitation (max)=492nm, Emission (max)=517nm. Store at -20°C. Please refer to protocols.