What is CFSE labeling?

What is CFSE labeling?

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.

What is CFSE proliferation assay?

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a compound that passively diffuses into cells. This compound proves useful when determining the ability of T cells to proliferate in response to antigen. Before antigen stimulation, previously frozen PBMC that have been thawed and rested are labeled with CFSE.

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.

What CFSE shows?

As controls, unstimulated CFSE-labeled CD8+ T cells, shows the CFSE intensity of non-divided cells, while the non-labeled cells shows the auto-fluorescence of the cells and the limits of detectable cell divisions.

Why is CFSE used?

Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent cell staining dye. Subsequent studies revealed that the dye can be used to monitor lymphocyte proliferation, both in vitro and in vivo, due to the progressive halving of CFSE fluorescence within daughter cells following each cell division.

Can you fix CFSE stained cells?

Cell proliferation dyes can be used to track cell divisions in vivo or in vitro. The staining can withstand fixation and permeabilization for subsequent immunostaining. Thus, cells labeled with CFSE can be subsequently fixed with formaldehyde or gluteraldehyde based fixatives.

How do you analyze cell proliferation?

Approaches to assess cell proliferation by flow cytometry include: measuring DNA synthesis with Invitrogen Click-iT EdU Flow Cytometry Assay Kits or traditional bromodeoxyuridine (BrdU) staining, tracking generations of cell division with Invitrogen CellTrace Cell Proliferation Kits or carboxyfluorescein succinimidyl …

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.

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.

Can you fix CFSE?

What is the labelling protocol for CFSE in vitro?

Anyway, the labelling protocol I use for in vitro proliferation assay is as follow: Incubate cells at the concentration of 10E6 cell/ml in PBS 1%FCS with 1uM CFSE (final concentration) for 10′ at 37°C, 5%CO2. Wash 2X in cold PBS 1%FCS (5′ at 1500 rpm and 4°C). Re-count the cells (you might lose some) and seed.

How is CFSE used in the cell cycle?

CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells.

How to check CFSE proliferation in T cells?

I seed my cells at 1 or 2×106/ml with 1 ml in a 24 well plate. they are stimulated with plate bound anti-cd3 and anti cd28. I take samples every day but only see proliferation after day 2.

Why do cells show decreased labeling with CFDA-SE?

If your cells show decreased labeling with the same stock of CFDA-SE, hydrolysis is the likely cause. Labeling concentration and conditions. Cells are usually labeled at a final CFDA-SE concentration of 0.5 to 5μM. The literature reports concentrations ranging from 0.2 to 10μM and even higher.