Fluorescent dye
Its mechanism is physicochemical: it relies on ubiquitous intracellular esterases (to generate fluorescence) and intracellular amines (for covalent binding). Consequently, any cell with active esterases (including most eukaryotic cells) can be labeled to track division or migration.

Preparation of CFDA-SE working solution
1.1 Prepare stock solution: it is recommended to dissolve 1 milligram CFDA-SE in 0.1794 mL DMSO to obtain 10 mM CFDA-SE.
Note: Store the stock solution at -20℃ or -80℃ in the dark and avoid repeated freezing.
1.2 Preparation of CFDA-SE working solution.
Dilute stock solution in serum-free cell culture.
Note: Please adjust the concentration of CFDA-SE working solution based on your specific needs.
Cell staining
2.1 Suspended cells: Centrifuge at 1000 g for 3-5 minutes at 4°C and discard the supernatant. Use PBS to wash twice, for five minutes each time.
Adherent cells: Discard the cell culture media, apply trypsin to separate the cells and produce a single cell suspension. Centrifuge at 1000 g for 3-5 minutes at 4°C and discard the supernatant. Use PBS to wash twice, for five minutes each time.
2.2 Add 1 mL CFDA-SE working solution and mix for 30 minutes.
2.3 Centrifuge at 400 g for 3-4 minutes at 4°C.
2.4 Wash the cells twice with PBS, five minutes/each time.
2.5 Resuspend the cells in serum-free medium or in PBS, and detect using fluorescence microscopy or flow cytometer.

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Cell proliferation labeling: CFSE can be used to label cells in vitro for proliferation assays. It freely diffuses into cells as a non - fluorescent form. Inside the cell, it is cleaved by cellular esterases, and the succinimidyl group irreversibly binds to intracellular amino groups, forming a fluorescent conjugate that remains in the cell. When cells divide, the fluorescent conjugate is evenly distributed to two daughter cells, and the fluorescence intensity of daughter cells is half of that of the parent cell. Flow cytometry can be used to detect the series - halved fluorescence intensity of CFSE - labeled cell proliferation populations [1]

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CFSE (carboxyfluorescein succinimidyl ester) is used as an intracellular fluorescent dye for tracking cell proliferation. In cell proliferation assays, CFSE fluorescence intensity (FI) within cells decreases over time due to natural decay and protein turnover, even in the absence of cell division. This decay exhibits a biphasic pattern and can be mathematically modeled. Experimental data from unstimulated PBMC cultures from two healthy donors showed a time-dependent decrease in mean CFSE FI over 160 hours. [1]
The study compared multiple mathematical models to describe the label decay kinetics. Models incorporating multiple loss rate mechanisms (e.g., separate rates for different fluorescent conjugates) or a single time-dependent decay rate (e.g., Gompertz decay model, \( \frac{dx}{dt} = -c(x - x_a)e^{-kt} \) ) provided better fits to the observed CFSE decay data compared to a simple exponential decay model. [1]
CFSE is used to track proliferating cells by flow cytometry, allowing discrimination of up to eight or more successive cell divisions. It has been applied to study division-related phenotypic and functional changes during differentiation of B cells, T cells, and hematopoietic precursor cells. It can also be combined with immunophenotyping using antibodies conjugated to PE, PerCP, or PE/Cy5. [2]

CFSE staining intensity is linear with respect to concentration, and fluorescence decays by about 60% in the first 1–2 days in non-dividing cells, then stabilizes for weeks to months. [2]

The dye has been used to examine slowly dividing glioblastoma cancer cells, erythroid progenitor proliferation, antigen-specific T cell clones, and neuroantigen-specific T cells in multiple sclerosis. [2]
In vitro, CFSE is primarily used to assess lymphocyte proliferation kinetics. Studies show that upon stimulation with cytokines like IL-2, CFSE-labeled human NK or T cells proliferate, demonstrating successive halving of CFSE fluorescence. This technique is highly sensitive, distinguishing proliferated progeny from non-proliferated precursors. It is also utilized in cytotoxicity assays (e.g., using a mix of CFSE-high and CFSE-low labeled target cells) to evaluate antigen-specific killing .

CFDA-SE is an intracellular and green fluorescent dye that may be used to detect strain colonization in vivo.
Method: For determine strain colonization in the intestine.
1. Strains are resuspended in PBS to obtain a cell density of 108 CFU/mL.
2. CFDA-SE (1 mM; 10 μL; 20 min; 37°C; dark/protect from light) is added to 1 mL of bacterial suspension.
3. Centrifuge (13000 g, 10 min, 4°C) and wash the mixture for 3 times using sterile PBS to remove excess CFDA-SE.
4. Strains are resuspended in sterile PBS (108 CFU/mL) and 1 mL of bacterial suspension is administered to SD rats. Day 1 and 3 after gavage, the fluorescence imaging is performed on anesthetized rats who are then sacrificed and different intestinal sections are taken for imaging.
5. The fluorescence imaging is performed using an IVIS Lumina III Smart Imaging System.

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- Cell tracking: In vivo, CFSE can remain in viable cells for several weeks at measurable concentrations, regardless of cell type or activation state. It can be used to track the division and proliferation of cells in the body, providing uniform labeling with little adverse effects on the cell's intracellular machinery [2]

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CFSE-labeled cells can be adoptively transferred in vivo (e.g., intravenous injection in mice) and tracked for several months. It has been used to study B cell division in the absence of T cell division, alloresponses in mixed lymphocyte reactions, and graft-versus-host response modulation. [2]

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In vivo, CFSE is widely used to track immune cell homing and proliferation, and to evaluate vaccine efficacy. For instance, antigen-specific target cells labeled with CFSE are adoptively transferred into immunized mice; the loss of CFSE signal indicates in vivo killing by CD8+ T cells. Additionally, intracerebroventricular injection of CFSE enables lineage tracing of specific cells like embryonic brain macrophages .

CFSE is primarily a cell labeling dye with limited cell-free applications. To verify dye properties: Dissolve CFSE in anhydrous DMSO (e.g., 5mM stock). For protein conjugation (if necessary), incubate CFSE with amine-containing protein in PBS (pH 7.4-8.0) for 30 min at room temperature in the dark. Remove unbound dye via centrifugation or dialysis. Excitation/emission maxima are ~492nm/517nm.

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second-generation dye, Cell Trace Violet (CTV), excited by 405-nm blue laser light. CTV is chemically related to CFSE, but allows the 488-nm line of the Argon laser to be used for other probes[2].

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Cell labeling and proliferation detection: Prepare cell suspensions, add an appropriate amount of CFSE solution, and incubate at an appropriate temperature for a certain period to allow CFSE to enter the cells. Then, wash the cells to remove unbound CFSE. Culture the labeled cells under appropriate conditions. At different time points, use flow cytometry to measure the fluorescence intensity of cells. According to the change of fluorescence intensity, analyze cell proliferation, division and other conditions [1]

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The standard procedure for CFSE labeling of cells involves using its precursor, carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE, due to its lipophilicity, passively diffuses across cell membranes and is taken up by cells. Once inside the cell, intracellular esterases (specifically, acetylesterase is hypothesized) catalyze the hydrolysis of its acetate esters, converting it into the highly fluorescent and membrane-impermeable CFSE. The succinimidyl ester moiety of CFSE then reacts with intracellular amine groups on proteins, forming stable fluorescent conjugates (e.g., CF-R2) that are retained, and unstable conjugates (e.g., CF-R1) that are lost or degraded. After initial exposure and staining, the cell culture is flushed to remove excess label. [1]
For the decay study, peripheral blood mononuclear cells (PBMCs) from two donors were stained with CFSE following the standard procedure but were not stimulated to divide. This ensured any observed fluorescence loss was due to natural decay processes. Cells were measured at 24 distinct time points over 160 hours in triplicate using flow cytometry, and the mean total fluorescence intensity (FI) of each sample was recorded. [1]
Cells are resuspended in PBS/0.1% BSA at 5 × 10⁷ cells/ml. CFSE is added to a final concentration of 10 µM and incubated for 10 minutes at 37°C. Staining is quenched with ice-cold RPMI 1640/10% FBS for 5 minutes on ice. Cells are washed three times in culture or injection medium. For proliferation assays, cells are cultured under appropriate conditions or adoptively transferred. Harvested cells can be stained with antibodies for immunophenotyping and analyzed by flow cytometry with excitation at 488 nm and emission collected with a 525-nm band-pass filter. [2]

For improved uniformity, CFSE can be diluted to 20 µM in PBS/0.1% BSA and added to an equal volume of 2× concentrated cell suspension. [2]

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CFSE dilution assay protocol: 1. Prepare single-cell suspension (e.g., PBMC) in pre-warmed PBS. 2. Add CFSE (final concentration 0.5-5µM) and incubate for 10-15 min at 37°C in the dark. 3. Quench with cold complete media (containing serum) and incubate on ice for 5 min. 4. Wash cells twice to remove excess dye. 5. Culture labeled cells with stimuli (e.g., anti-CD3/CD28, IL-2). 6. Harvest after 3-7 days for flow cytometry; analyze proliferation index via halving of fluorescence intensity .

For adoptive transfer, CFSE-labeled cells (e.g., 1 × 10⁷ to 5 × 10⁷ cells per mouse) are injected intravenously via the tail vein in mice or rats. Cells are tracked in lymphoid organs such as the spleen, and analyzed by flow cytometry at various time points post-transfer. [2]

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In Vivo CTL Assay Protocol: 1. Label target cells (splenocytes) with low (0.5µM) or high (5µM) CFSE. 2. Pulse the high-CFSE population with specific antigen peptide; keep low-CFSE population as control (unpulsed). 3. Mix cells at 1:1 ratio. 4. Adoptively transfer the mixture into immunized recipient mice via tail vein or retro-orbital injection. 5. Harvest (4-24h post-transfer), isolate splenocytes, and analyze CFSE signal by flow cytometry. Specific killing results in loss/reduction of the peptide-pulsed (high-CFSE) peak .

The fluorescence intensity of CFSE decreases by about 60% in the first 1-2 days in non-dividing cells due to the catabolism of unstable binding components, and then remains stable for several weeks to months. [2] The dye has a long half-life in vivo, allowing for monitoring for several months after transplantation. [2]

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The pharmacokinetics of CFSE depend heavily on the labeled cells. Free CFSE hydrolyzes rapidly in aqueous solutions like plasma (very short half-life). Once loaded into cells, it covalently binds via succinimidyl groups to intracellular proteins, becoming highly stable. In non-dividing cells, CFSE retention lasts for weeks. During cell division, fluorescence intensity decreases logarithmically proportional to division number. Clearance in vivo reflects the turnover rate of labeled cells .

High concentrations of CFSE can affect cell viability and division. Optimal staining results should increase fluorescence intensity by 3-3.5 log units relative to unstained cells. Adding protein (0.1% BSA) during staining can improve cell viability and reduce cell aggregation. Staining at lower temperatures (on ice or at room temperature) may also improve cell viability. [2]
CFSE can alter the expression of certain cell surface markers or reduce the staining effect of certain monoclonal antibodies, possibly due to steric hindrance. [2]

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CFSE exhibits concentration-dependent cytotoxicity. High concentrations or prolonged exposure can impair cell function, induce activation-induced cell death (AICD), or inhibit proliferation. To balance labeling efficiency and viability, it is crucial to optimize staining conditions (e.g., lower concentrations 0.5-2µM) and perform thorough washing post-labeling. For primary lymphocytes, quenching with high-serum media effectively reduces toxicity .

[1]. Quantifying CFSE Label Decay in Flow Cytometry Data. Appl Math Lett. 2013 May 1;26(5):571-577.

[2]. Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr Protoc Cytom. 2013;Chapter 9:Unit9.11.

[3]. Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization. J Cell Mol Med. 2021 Jun;25(12):5586-5601.

[4]. Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment. Oncoimmunology. 2017; 6(7): e1333210.

We developed a series of label decay models for cell proliferation experiments using the intracellular dye carboxyfluorescein succinimide (CFSE) as a staining agent. We validated these models using data collected from two healthy patients and compared them with the Akiake Information Criterion (AIC). The characteristics of various decay rates in the data were characterized and interpreted by time-dependent decay models, such as the logistic model and the Gompertz model. [1] Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease, and despite the many successes of immunotherapy in the treatment of other malignancies in recent years, the cure rate remains low. Given the large infiltration of effector T cells in human diseases, we hypothesized that accurately mimicking the immune microenvironment of PDA would help us study mechanisms of immune suppression that can be overcome, thereby gaining therapeutic benefits. To this end, we developed an organoid culture system using in vivo precisely cut sections of fresh PDA. We demonstrated that cultured pancreatic sections retained their baseline morphology, surface area, and microenvironment after at least 6 days of culture, and section viability was confirmed by MTT assay and immunohistochemical staining with Ki-67 and cleaved-Caspase-3 antibodies. Immune cells, including T cells (CD3+, CD8+, and FOXP3+), macrophages (CD68+, CD163+, and HLA-DR+), and stromal myofibroblasts (αSMA+), were present throughout culture. Comprehensive proteomic analysis of pancreatic ductal adenocarcinoma (PDA) sections before and after culture showed that most identified immune proteins remained stable during culture. Cytotoxic effects of drugs (astrosporin, STS, and cyclohexylimide, CHX) on PDA section culture confirmed that this system can be used to assess functional response and cell viability after drug treatment, and that this assessment is treatment- and dose-dependent. We used multicolor immunofluorescence to stain live sections to detect cancer cells (EpCAM+) and immune cells (CD11b+ and CD8+). Finally, we confirmed that autologous CFSE-labeled spleen cells could rapidly migrate into co-cultured tumor sections. Therefore, this study shows that tumor section culture can be used to study the immune microenvironment of pancreatic ductal adenocarcinoma (PDA). [4]

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CFSE is a colorless, non-fluorescent, nonpolar molecule. It has a succinimide group that can specifically bind to cells and a carboxyfluorescein diacetate group that can be non-enzymatically hydrolyzed. It is a good live cell marker. In immunological experiments, the use of CFSE-labeled cells can replace traditional morphological observation and 3H-TdR incorporation methods, thereby improving the research level and expanding the application of new immunological technologies [2].

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CFSE is the active fluorescent form generated in cells by its precursor CFDA-SE. It is an important tool for mapping cell division history in proliferation experiments. After CFSE covalently binds to long-lived intracellular proteins (forming CF-R2), the fluorescent label can be retained in live cells for several weeks, thereby achieving long-term tracking. The decrease in fluorescence intensity over time (label decay) is due to the natural decay of the dye and the turnover of the proteins it binds to [1]. The main application discussed in this paper is the use of CFSE in flow cytometry-based cell proliferation assays. CFSE fluorescence is serially diluted with each cell division to establish a correlation between the measured fluorescence intensity and the number of cell divisions. Accurate simulation of the natural decay of CFSE fluorescence is crucial for correctly estimating cell proliferation and mortality based on such data. [1] Mathematical models used in the literature to quantify CFSE label decay include sets of ordinary differential equations representing the conversion and decay of different intracellular fluorescent substances (CFDA-SE, CFSE, CF-R1, CF-R2), as well as simplified single-equation models using exponential, logistic, or Gompertz decay kinetics. The Gompertz decay model has a time-dependent decay rate and fits the observed biphasic decay data more accurately than the simple exponential model. [1]CFSE (carboxyfluorescein diacetate succinimide ester) is a cell-permeable fluorescein dye that covalently binds to intracellular amino groups, thereby distributing evenly to daughter cells during cell division. It is excited at 488 nm and emits at approximately 525 nm, and is compatible with fluorescein filter sets. [2]
The second-generation dye Cell Trace Violet (CTV) is excited at 405 nm, has similar cell division tracking capabilities, and allows the 488 nm channel to be used for other probes. [2]
CFSE data can be used in modeling software (such as ModFit) to calculate precursor cell frequency and proliferation index. [2]

C29H19NO11

557.461268663406

557.095

C, 62.48; H, 3.44; N, 2.51; O, 31.57

150347-59-4

Off-white to yellow solid powder

1.6±0.1 g/cm3

757.9±70.0 °C at 760 mmHg

152-154ºC(lit.)

412.2±35.7 °C

0.0±2.6 mmHg at 25°C

1.701

0.5

C12(OC(=O)C3=CC(C(=O)ON4C(=O)CCC4=O)=CC=C13)C1C=CC(OC(=O)C)=CC=1OC1C=C(OC(=O)C)C=CC2=1

JGPOSNWWINVNFV-UHFFFAOYSA-N

InChI=1S/C29H19NO11/c1-14(31)37-17-4-7-20-23(12-17)39-24-13-18(38-15(2)32)5-8-21(24)29(20)22-11-16(3-6-19(22)28(36)40-29)27(35)41-30-25(33)9-10-26(30)34/h3-8,11-13H,9-10H2,1-2H3

(2,5-dioxopyrrolidin-1-yl) 3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylate

5(6-Carboxyfluorescein diacetate succinimidyl ester; CFDA-SE; 5(6-CFDA N-succinmidyl ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~89.69 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)