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CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*

Treponema pallidum-platelet interactions. (A) Flow cytometry isolates the co-localized platelet and treponeme populations by SSC and FSC gating. Dot plots of (B) CFSE-labeled heat-treated treponemes demonstrate reduced binding to human platelets labeled with PE/Cy5 anti-CD41a compared with (C) CSFE-labeled viable treponemes. (D) CFSE-labeled viable treponemes bound significantly more human platelets (mean = 55.13% ± 2.91 [SD] *P<0.0001) compared with heat-treated treponemes (mean = 19.05% ± 2.29 [SD]) following co-incubation for 16 hours at 37°C and ~ 5% oxygen. Results represent the mean of three independent experiments with statistical significance computed by two-way ANOVA, with a minimum of 3 replicates per sample type per experiment. (E) Darkfield microscopy FOV counts (20 random locations/slide) demonstrate viable treponemes bind significantly more human platelets (mean = 31.73% ± 1.19 [SD] *P<0.0001) than heat-treated treponemes (mean = 6.47% ± 1.19 [SD]) following co-incubation at 5% oxygen at 34°C for 48 hours. Results represent the mean of three independent experiments with statistical significance computed by unpaired two-tailed Student’s t test, n = 3. (F) Heat-treated treponemes (top) are non-motile yet morphologically similar to viable treponemes (bottom). (G) Darkfield microscopy at 1000x magnification demonstrates platelet interactions are reversible. Image capture from video micrographs show edgewise attachment of a treponeme to an activated platelet (0.01 s to 8.10 s) which then detaches and moves away (9.05 s). Source: <strong>Interaction of Treponema pallidum, the syphilis spirochete, with human platelets </strong>by Church et al., <em>PLOS</em>,  Jan. 2019.
Treponema pallidum-platelet interactions. (A) Flow cytometry isolates the co-localized platelet and treponeme populations by SSC and FSC gating. Dot plots of (B) CFSE-labeled heat-treated treponemes demonstrate reduced binding to human platelets labeled with PE/Cy5 anti-CD41a compared with (C) CSFE-labeled viable treponemes. (D) CFSE-labeled viable treponemes bound significantly more human platelets (mean = 55.13% ± 2.91 [SD] *P<0.0001) compared with heat-treated treponemes (mean = 19.05% ± 2.29 [SD]) following co-incubation for 16 hours at 37°C and ~ 5% oxygen. Results represent the mean of three independent experiments with statistical significance computed by two-way ANOVA, with a minimum of 3 replicates per sample type per experiment. (E) Darkfield microscopy FOV counts (20 random locations/slide) demonstrate viable treponemes bind significantly more human platelets (mean = 31.73% ± 1.19 [SD] *P<0.0001) than heat-treated treponemes (mean = 6.47% ± 1.19 [SD]) following co-incubation at 5% oxygen at 34°C for 48 hours. Results represent the mean of three independent experiments with statistical significance computed by unpaired two-tailed Student’s t test, n = 3. (F) Heat-treated treponemes (top) are non-motile yet morphologically similar to viable treponemes (bottom). (G) Darkfield microscopy at 1000x magnification demonstrates platelet interactions are reversible. Image capture from video micrographs show edgewise attachment of a treponeme to an activated platelet (0.01 s to 8.10 s) which then detaches and moves away (9.05 s). Source: <strong>Interaction of Treponema pallidum, the syphilis spirochete, with human platelets </strong>by Church et al., <em>PLOS</em>,  Jan. 2019.
Effects of GTM (1, 1/2 and 1/4 -fold serum concentrations) and probenecid (Prob, 80 μM) on the intracellular accumulation of 6-CF in CHO-hOAT1 cells (left) and 5-CF in HEK293-hOAT3 cells.(right). The effects of GTM on the uptake activity of hOAT1 and hOAT3 are shown in Fig. 6. GTM at 1/4−, 1/2− and 1-fold serum concentrations significantly reduced the intracellular accumulation of 6-CF, an OAT1 substrate, by 43.9, 41.9 and 58.1%, respectively, when compared with blank serum specimen at corresponding concentrations. Likewise, GTM at 1/2− and 1-fold serum concentration significantly reduced the intracellular accumulation of 5-CF, an OAT3 substrate, by 36.4% and 31.3%, respectively, when compared to blank serum specimen at corresponding concentration. As positive control inhibitors of hOAT1 and hOAT3, probenecid (80 μM) significantly reduced the intracellular accumulation of 6-CF and 5-CF by 50.4 and 50.7%, respectively. These <em>in vitro</em> studies indicated that GTM significantly inhibited the uptake transport mediated by hOAT1 and hOAT3. *P < 0.05, **P < 0.01 and ***P < 0.001. Source: <strong>Green tea inhibited the elimination of nephro-cardiovascular toxins and deteriorated the renal function in rats with renal failure </strong>by Peng et al.,<em> Scientific Reports</em>, Nov. 2015.
Chemical structure for CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4*
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Physical properties
Molecular weight557.46
SolventDMSO
Spectral properties
Excitation (nm)498
Emission (nm)517
Storage, safety and handling
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

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CAS
150347-59-4
Molecular weight
557.46
Excitation (nm)
498
Emission (nm)
517
It is widely recognized that fluorescent labeling of cells is an effective means to determine total cell numbers or how many viable cells exist in a sample. Flow cytometry combined with fluorescent staining is a powerful tool to analyze heterogeneous cell populations. Fluorescein diacetate (FDA) and its derivatives are non-fluorescent molecules that diffuse into cells and are hydrolyzed by intracellular non-specific esterases to give fluorescent products. The fluorescent products can be accumulated only in those cells that have intact cell membranes; therefore, dead cells with leaky membranes are not stained. The precise kinetics of membrane transport and intracellular hydrolysis of FDA and its analogs (such as CDCFDA) are related to cellular functions, thus FDA labeling can be used for monitoring cells by flow cytometry or fluorescence microscopy. The fluorescence intensity of labeled cells by FDA dyes varies considerably among cell lines and strains, probably because of differences in intracellular esterase activity. CFSE is an amine-reactive FDA derivative that is widely used for monitoring cell proliferation with a flow cytometer.

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Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester] *CAS 150347-59-4* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM179.385 µL896.925 µL1.794 mL8.969 mL17.939 mL
5 mM35.877 µL179.385 µL358.77 µL1.794 mL3.588 mL
10 mM17.939 µL89.693 µL179.385 µL896.925 µL1.794 mL

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Spectral properties

Excitation (nm)498
Emission (nm)517

Citations


View all 6 citations: Citation Explorer
Interaction of Treponema pallidum, the syphilis spirochete, with human platelets
Authors: Church, Brigette and Wall, Erika and Webb, John R and Cameron, Caroline E
Journal: PloS one (2019): e0210902
Antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B lymphocytes enhances the killing effect of cytotoxic T lymphocytes on tumor stem-like cells derived from cisplatin-resistant lung cancer cells
Authors: Zhang, Xueyan and Zhang, Yanwei and Xu, Jianlin and Wang, Huimin and Zheng, Xiaoxuan and Lou, Yuqing and Han, Baohui
Journal: Journal of Cancer (2018): 367
Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel
Authors: Xu, Yufan and Wang, Xiaohong
Journal: Biotechnology and bioengineering (2015): 1683--1695
Overexpression of the CaTIP1-1 pepper gene in tobacco enhances resistance to osmotic stresses
Authors: Yin, Yan-Xu and Wang, Shu-Bin and Xiao, Huai-Juan and Zhang, Huai-Xia and Zhang, Zhen and Jing, Hua and Zhang, Ying-Li and Chen, Ru-Gang and Gong, Zhen-Hui
Journal: International journal of molecular sciences (2014): 20101--20116
Identification of VAR2CSA domain-specific inhibitory antibodies of the Plasmodium falciparum erythrocyte membrane protein 1 using a novel flow cytometry assay
Authors: Obiakor, Harold and Avril, Marion and MacDonald, Nicholas J and Srinivasan, Prakash and Reiter, Karine and Anderson, Charles and Holmes, Kevin L and Fried, Michal and Duffy, Patrick E and Smith, Joseph D and others, undefined
Journal: Clinical and Vaccine Immunology (2013): 433--442
Aging enhances maceration-induced ultrastructural alteration of the epidermis and impairment of skin barrier function
Authors: Minematsu, Takeo and Yamamoto, Yuko and Nagase, Takashi and Naito, Ayumi and Takehara, Kimie and Iizaka, Shinji and Komagata, Kazunori and Huang, Lijuan and Nakagami, Gojiro and Akase, Tomoko and others, undefined
Journal: Journal of dermatological science (2011): 160--168

References


View all 68 references: Citation Explorer
Novel method for cell debris removal in the flow cytometric cell cycle analysis using carboxy-fluorescein diacetate succinimidyl ester
Authors: Terho P, Lassila O.
Journal: Cytometry A (2006): 552
Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods
Authors: Brouwer N, Kohen J, Jamie J, Vemulpad S.
Journal: J Microbiol Methods (2006): 234
Optimisation of the fluorescein diacetate antibacterial assay
Authors: Wan, undefined and y S, Brouwer N, Liu Q, Mahon A, Cork S, Karuso P, Vemulpad S, Jamie J.
Journal: J Microbiol Methods (2005): 21
A three-dimensional flow control concept for single-cell experiments on a microchip. 2. Fluorescein diacetate metabolism and calcium mobilization in a single yeast cell as stimulated by glucose and pH changes
Authors: Peng XY, Li PC.
Journal: Anal Chem (2004): 5282
Effect of immunosuppressants on T-cell subsets observed in vivo using carboxy-fluorescein diacetate succinimidyl ester labeling
Authors: Hu H, Dong Y, Feng P, Fechner J, Hamawy M, Knechtle SJ.
Journal: Transplantation (2003): 1075
Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry
Authors: Drobni P, Mistry N, McMillan N, Ev and er M., undefined
Journal: Virology (2003): 163
Detection of Saccharomyces cerevisiae carboxylesterase activity after native and sodium dodecyl sulfate electrophoresis by using fluorescein diacetate as substrate
Authors: Lomolino G, Lante A, Crapisi A, Spettoli P, Curioni A.
Journal: Electrophoresis (2001): 1021
Fluorescein diacetate hydrolysis as a measure of fungal biomass in soil
Authors: Gaspar ML, Cabello MN, Pollero R, Aon MA.
Journal: Curr Microbiol (2001): 339
Extrusion of fluorescein diacetate by multidrug-resistant Candida albicans
Authors: Yang HC, Mikami Y, Imai T, Taguchi H, Nishimura K, Miyaji M, Branchini ML.
Journal: Mycoses (2001): 368
Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity
Authors: Clarke JM, Gillings MR, Altavilla N, Beattie AJ.
Journal: J Microbiol Methods (2001): 261