Cell Function
Cell Functions | Comprehensive Guide of the Principles and Precautions of CFSE Detection of Cell Proliferation
Source: Elabscience®Published: Oct 16,2024
Cell proliferation is the process by which cells divide, involving complex reactions such as DNA replication, transcription, and protein synthesis. This process is crucial for the physiological functioning of living cells. Monitoring cell proliferation is important for assessing cellular health, metabolic balance, disease conditions, and cell viability. CFSE (carboxyfluorescein succinimidyl ester) is a long-term cell tracer dye that can fluorescently label cells. It offers advantages over other proliferation detection methods, such as the MTT assay or [³H]-thymidine incorporation, including better stability, sensitivity, and lower toxicity. Today, we will explain the principles and operational precautions for using CFSE in cell proliferation detection.
Principles of CFSE Detection
CFSE, also known as CFDA SE, is a membrane-permeable fluorescent dye that is non-fluorescent by itself. Once it penetrates the cell membrane and enters the living cell, intracellular esterases catalyze it into carboxyfluorescein succinimidyl ester (CFSE), which emits strong green fluorescence and is retained within the cell as it cannot cross the cell membrane. CFSE spontaneously and irreversibly binds to amine groups on intracellular proteins, thereby coupling to these proteins. Excess, unbound CFSE diffuses out of the cell into the culture medium and is removed by subsequent washing steps.
CFSE-labeled cells exhibit uniform and stable fluorescence. During cell division, the fluorescence is equally distributed to the two daughter cells, with the fluorescence intensity halved. Flow cytometry can detect and analyze cells based on their fluorescence intensity, distinguishing between non-divided cells and cells that have undergone one (1/2 intensity), two (1/4 intensity), three (1/8 intensity), or more divisions. CFSE can detect up to eight or more cell divisions. CFSE-labeled cells can be used for both in vitro and in vivo proliferation studies without staining neighboring cells. CFSE-labeled cells exhibit green fluorescence ,Ex=494 nm，Em=521 nm, detectable by flow cytometry or fluorescence microscopy.
CFSE Detection Protocol and Precautions
CFSE detection is commonly used for both cell line samples and primary cell samples. The protocol and precautions are as follows:
| Staining Procedure |
Precautions |
|---|---|
|
Reagent Preparation |
CFSE powder is relatively stable. After preparing CFSE stock solution, aliquot and store it in a sealed, light-protected manner at -20℃, and use within one month. CFDA SE is prone to hydrolysis and degradation in aqueous solution; therefore, store it sealed. |
|
Cell Staining - Cell Lines and Primary Cells |
Use PBS as the staining dilution buffer. Azides, serum, or proteins in culture medium or other buffers may bind free dye and interfere with cell staining. Adjust cell density to 1 × 10⁷ cells/mL (optimal staining density). Incubate cells for staining in a 37℃ cell culture incubator, protected from light, for no more than 10 minutes for cell lines and no more than 5 minutes for primary cells. Recommended CFSE staining concentration is 2.5~5 μM. |
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Other Details |
Cell washing involves multiple centrifugation steps. To minimize cell loss due to centrifugation, adjust the centrifuge settings so that the acceleration does not exceed 3 and the deceleration does not exceed 1 (Acc ≤ 3, Dec ≤ 1). CFSE can solidify at lower temperatures, adhering to the bottom, walls, or cap of the centrifuge tube. After rewarming and dissolving, centrifuge gently and mix thoroughly before use. |
Figure 1. illustrates in vitro induced mouse bone marrow-derived dendritic cells co-cultured for 72 hours with normal mouse spleen cells (stained with E-CK-A345 (CFSE)) after LPS-induced maturation, followed by co-staining with Elab Fluor® Red 780 Anti-Mouse CD8a (E-AB-F1104S) and APC Anti-Mouse CD4 (E-AB-F1097E), and subsequent analysis of T cell proliferation using flow cytometry.
The above details the principles and operational precautions for using CFSE in cell proliferation detection. As an efficient and sensitive cell proliferation tracking tool, CFSE provides researchers with a simple and reliable method for monitoring cell division.
