5 and 6 carboxyfluorescein diacetate succinimidyl ester cfse

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5-and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) is a fluorescent dye used for cell labeling and tracking. It is a cell-permeant compound that becomes fluorescent upon hydrolysis by intracellular esterases and covalent binding to cellular proteins.

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Lab products found in correlation

Concanavalin a (cona), by Merck Group (3 mentions) Far red, by Thermo Fisher Scientific (2 mentions) Tnf α, by Thermo Fisher Scientific (2 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (2 mentions) 2 4 6 trinitrobenzenesulfonic acid, by Merck Group (2 mentions) Goat anti mouse igg hrp, by Southern Biotech (2 mentions) Goat anti mouse igm hrp, by Southern Biotech (2 mentions) Magnetic anti goat mouse ig microbeads, by Miltenyi Biotec (2 mentions) Rgg and bsa, by Merck Group (2 mentions) Whole chicken ovalbumin ova, by Merck Group (2 mentions) Elispot kit, by BD (2 mentions) Nigericin, by Merck Group (1 mentions) Sheep anti mouse igg hrp, by Cytiva (1 mentions) Anti human caspase 1, by Santa Cruz Biotechnology (1 mentions) Anti mouse il 1β, by R&D Systems (1 mentions) Alexa fluor 594conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions) Goat anti rabbit igg horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions) Anti gapdh, by Santa Cruz Biotechnology (1 mentions) Anti α tubulin, by Santa Cruz Biotechnology (1 mentions) Cytochalasin d, by Merck Group (1 mentions)

Spelling variants of this product

CFSE (2779 citations) Carboxyfluorescein diacetate succinimidyl ester (CFSE) (55 citations) Carboxyfluorescein diacetate succinimidyl ester (44 citations) 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (19 citations) 5,6-carboxyfluorescein diacetate succinimidyl ester (9 citations) 5-(and -6)-CFSE (4 citations) 5-(and-6)-carboxyfluorescein (3 citations) 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (2 citations) 5(6)-Carboxyfluorescein (2 citations)

23 protocols using 5 and 6 carboxyfluorescein diacetate succinimidyl ester cfse

Neutrophil Adhesion Dynamics Under Shear

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To examine neutrophil adherence to endothelial cells under shear stress, mouse endothelial cells (Wang, et al., 2008 (link)) were cultured to confluency on 10ng/ml fibrinogen (Fn) coated coverslips and treated with 50 ng/ml TNFα (Peprotech) for 4 hours at 37°C. The coverslips containing the endothelial ce ll layer were washed with PBS and placed in a flow chamber apparatus (GlycoTech). The WT and Srgap2 deficient neutrophils labeled with 1 μM CFSE (5-[and 6]-carboxyfluorescein diacetate succinimidyl esters) (Thermo Fisher Scientific) and 1 μM Far-Red (Thermo Fisher Scientific) respectively were mixed at 1:1 ratio, and flowed into the chamber at a shear flow rate of 1 dyn/cm². The adherent cells were then examined and counted under a fluorescence microscope. We alternated the labeled group in the study to completely eliminate the possibility of any influence from the dye.
To examine polarization in neutrophils adhered to polylysine (PK)-coated coverslips under shear stress, neutrophils were allowed to sediment to the coverslips for 10 min in the flow chamber. The sheer stress was gradually ramped up to 2 dyn/cm² for 10 min before being imaged by a confocal microscope.

Ren C., Yuan Q., Braun M., Zhang X., Petri B., Zhang J., Kim D., Guez-Haddad J., Xue W., Pan W., Fan R., Kubes P., Sun Z., Opatowsky Y., Polleux F., Karatekin E., Tang W, & Wu D. (2019). Leukocyte cytoskeleton polarization is initiated by plasma membrane curvature from cell attachment. Developmental cell, 49(2), 206-219.e7.

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Neutrophil Adhesion and Polarization under Shear Stress

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To examine neutrophil adherence to endothelial cells under shear stress, mouse endothelial cells (50 (link)) were cultured to confluency on 10ng/ml fibrinogen (Fb) coated coverslips and treated with 50 ng/ml TNFα (Peprotech) for 4 hours at 37°C. The coverslips containing the endothelial cell layer were washed with PBS and placed in a flow chamber apparatus (GlycoTech). The siCtr and siARF6 transfected neutrophils were labeled with 1 μM CFSE (5-[and 6]-carboxyfluorescein diacetate succinimidyl esters) (Thermo Fisher Scientific) and 1 μM Far-Red (Thermo Fisher Scientific) respectively, and mixed at 1:1 ratio, then flowed into the chamber at a shear flow rate of 1 dyn/cm². The adherent cells were then examined and counted under a widefield fluorescence microscope. We alternated the labeled group in the study to eliminate the possibility of any influence from the dye.
To examine polarization in neutrophils that are adhered to endothelial cells under shear stress, neutrophils were flowed through the chamber seeded with mouse endothelial cells pretreated with TNFα at a shear flow rate of 1 dyn/cm² and imaged by a confocal microscope.

Ren C., Yuan Q., Jian X., Randazzo P.A., Tang W, & Wu D. (2020). Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization. The Journal of Immunology Author Choice, 204(4), 1012-1021.

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Purification and CFSE Labeling of Naïve CD4 T-Cells

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Purified T-cell populations were obtained from lymph nodes using a magnetic bead/column system (MACS; Miltenyi Biotech). Naïve CD4-SP T-Cells were isolated by first labelling total lymph node cells with anti-B220 biotin, anti-CD11b biotin, anti-CD8 biotin, anti-CD44 biotin and anti-CD25 biotin. CD4-SP cells (unlabelled fraction) were isolated by magnetic columns after being washed and labelled with strepavidin microbeads. Purity was >90%. Tetramer-mediated cell enrichment was performed as described30 (link). For CFSE (5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, Molecular Probes) labelling, cells were incubated with 5μg ml⁻¹ of CFSE for 15 min at 37 °C. Cell division was assessed by determining the percentage of CFSE^lo/med cells by flow cytometry and dividing by the percentage of CFSE^lo/med cells obtained following stimulation with PMA and ionomycin.

Hwang S., Palin A.C., Li L., Song K.D., Lee J., Herz J., Tubo N., Chu H., Pepper M., Lesourne R., Zvezdova E., Pinkhasov J., Jenkins M.K., McGavern D, & Love P.E. (2015). TCR ITAM multiplicity is required for the generation of follicular helper T-cells. Nature Communications, 6, 6982.

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Isolation and CFSE Labeling of Naive CD4+ T Cells

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Purified T cell populations were obtained from lymph nodes using a magnetic bead/column system (MACS; Miltenyi Biotech). Naïve CD4⁺ T Cells were isolated by first labeling total lymph node cells with anti-B220 biotin, anti-CD11b biotin, anti-CD8 biotin, anti-CD44 biotin, and anti-CD25 biotin. CD4⁺ cells (unlabeled fraction) were isolated by magnetic columns after being washed and labeled with strepavidin microbeads. Purity was >90%. Tetramer-mediated cell enrichment was performed as described ^{30 (link)}. For CFSE labeling, cells were incubated with 5 ug/ml of CFSE (5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, Molecular Probes) for 15 minutes at 37^oC. Cell division was assessed by determining the percentage of CFSE^lo/med cells by FACS and dividing by the percentage of CFSE^lo/med cells obtained following stimulation with PMA and ionomycin.

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Inflammasome Activation Assay Protocol

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PMA (cat# P1585), ATP (cat# A7699), nigericin (cat# N7143), 4′,6-diamidino-2-phenylindole (DAPI; cat# D9542), and cytochalasin D (cat# C8273) were purchased from Sigma-Aldrich (St Louis, MO, USA), anti-ASC (cat# SC-22514-R), anti-human caspase-1 (cat# SC-56036), anti-mouse caspase-1 (cat# SC-514), anti-human IL-1β (cat# SC-32294), anti-α-tubulin (cat# SC-32293), anti-GAPDH (cat# SC-32233), and goat anti-rabbit IgG-horseradish peroxidase (HRP; cat# SC-2004) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-NLRP3 (cat# AG-20B-0014) from Adipogen (San Diego, CA, USA), anti-mouse IL-1β (cat# AF-401-NA) from R&D Systems Inc. (Minutesneapolis, MN, USA); sheep anti-mouse IgG-HRP (cat# NA931) from Amersham (Amersham, UK), (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; cat# C1157) and Alexa Fluor-594 conjugated goat-anti-mouse IgG (H+L) (cat# A-11005) from Invitrogen (Carlsbad, CA, USA); and fluoresbrite yellow green carboxylate microspheres (1-μm YG beads, cat# 15702) from Polysciences Inc. (Warrington, PA, USA).

Chung I.C., OuYang C.N., Yuan S.N., Lin H.C., Huang K.Y., Wu P.S., Liu C.Y., Tsai K.J., Loi L.K., Chen Y.J., Chung A.K., Ojcius D.M., Chang Y.S, & Chen L.C. (2019). Pretreatment with a Heat-Killed Probiotic Modulates the NLRP3 Inflammasome and Attenuates Colitis-Associated Colorectal Cancer in Mice. Nutrients, 11(3), 516.

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In Vitro Cytotoxic T-Cell Assay

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The in vitro CTL assay was performed as previous described (21 (link),24 (link)). The splenocytes from immunized mice were re-stimulated with 2μg/ml of AFP₂₁₂ or AFP₄₉₉ peptide for indicated times in the RPMI media containing 20 units/ml of IL-2. The EL4 and EL4-AFP tumor cells were labeled with 1μM and 0.03μM of 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen), respectively, for 10min at 37°C. After washing, equal numbers (5×10⁴) of CFSE^lo-labeled target EL4-AFP cells and CFSE^hi-labeled EL4 control cells were co-cultured in triplicate with the in vitro peptide-stimulated splenocytes at the indicated E/T ratios for 6 hours. The killing of target EL4-AFP cells was analyzed by flow cytometry. The specific killing activity was calculated using the formula [1-(ratio of CFSE^hi/CFSE^lo in the absence of CTL) / (ratio of CFSE^hi/CFSE^lo in the presence of CTL)] ×100 as previously described (21 (link)).

Wu S., Zhu W., Peng Y., Wang L., Hong Y., Huang L., Dong D., Xie J., Merchen T., Kruse E., Guo Z.S., Bartlett D., Fu N, & He Y. (2017). The antitumor effects of vaccine-activated CD8+ T cells associate with weak TCR signaling and induction of stem-like memory T cells. Cancer immunology research, 5(10), 908-919.

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Modulation of Dendritic Cell Activation

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ConA and lipopolysaccharide (LPS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against CD11C (Cat #97585) and Beclin-1 (Cat #3495) were purchased from Cell Signaling Technology (Danvers, MA, USA). LC3B (sc-376404), ATG7 (sc-376212) and HLA-DR (53319) were purchased from Santa Cruz Biotechnology (Europe), and β-actin was purchased from ZSGB-BIO (Beijing, China). MHC-II (abs 125192) was purchased from Absin (Shanghai, China). P62 (R1309-8) was purchased from HUABIO (Hangzhou, China). ELISA kits were from MultiSciences (Hangzhou, China). Anti-CD4 microbeads and fluorochrome-coupled antibodies for CD11C, CD4, CD3, HLA-DR, CD69, MHC-II, CD80 and CD86 were from Biolegend (San Diego, CA, USA). Mouse recombinant GM-CSF, IL-4, tumor necrosis factor-α (TNF-α), and IL-33 were obtained from Novoprotein (China). Additionally, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Invitrogen (San Diego). Mouse lymphocyte separation medium (7211011) was obtained from DAKEWE (Shenzhen, China). 3-MA was purchased from Selleck (Houston, TX, USA). Bafilomycin A1 was from MedChemExpress (NJ, USA).

Fan X., Men R., Huang C., Shen M., Wang T., Ghnewa Y., Ma Y., Ye T, & Yang L. (2020). Critical roles of conventional dendritic cells in autoimmune hepatitis via autophagy regulation. Cell Death & Disease, 11(1), 23.

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Immunomodulatory effects of compounds

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Con A, chloroquine, and DHA were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), including the antibodies against p62 (Cat# 5114), LC3 (D11), phospho-STAT1 (58D6), STAT1 (D1K9Y), phospho-STAT3 (D3A7), STAT3 (D3Z2G), phospho-NF-κB p65 (93H1), NF-κB p65 (C22B4), and GAPDH (D16H11). Mouse monoclonal antibodies against CD3 (145-2C11), CD4 (RM4-5), IFN-γ (XMG1.2), and CD69 (H1.2F3) were purchased from BD Pharmingen (San Jose, CA, USA). Anti-NK1.1 (PK136), anti-CD16/CD32 (2.4G2), goat anti-rabbit IgG, and 7-aminoactinomycin D (7-AAD) were from MultiSciences (Hangzhou, China). Also, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Invitrogen (San Diego, CA, USA).

Li Y., Tang Y., Wang S., Zhou J., Zhou J., Lu X., Bai X., Wang X.Y., Chen Z, & Zuo D. (2016). Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation. Frontiers in Immunology, 7, 350.

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BCG-Induced Immune Cell Trafficking

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Animals were inoculated in the hind footpad with 1x10⁶ CFUs of BCG in 30 μl of PBS. Control animals received an equal volume of PBS. For assessment of cell migration from the footpad skin, animals previously injected with BCG or PBS where injected 24hrs before sacrifice in the same footpad with 20 μl of 0.5 mM 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). In select experiments animals were injected with 50 ng or 300 ng rTNF-α or 2 μg rIL-12p40 homodimer (R&D Systems) in the same footpad that 2hrs earlier was inoculated with BCG or PBS. For assessment of lymphatic drainage to LNs, 20 μl 5% Evan’s blue (Sigma) was injected in the footpad and LNs isolated at different time points after injection. For assessment of CD4⁺ antigen-specific T-cell responses, 1x10⁵ LN cells from P25 TCRTg RAG–1^-/- mice were first labeled with 1 μM CFSE and then injected i.v. in the tail vein of congenic CD45.1⁺ recipients in a final volume of 200 μl. For DC adoptive transfer experiments, 2x10⁶ naïve BMDCs generated from WT or transgenic mice were labeled with 3 μM CFSE and then injected into the footpad in a final volume of 40 μl. Two hrs after DC transfer, the same footpads were inoculated with 30 μl PBS or 1x10⁶ CFUs of BCG.

Bollampalli V.P., Harumi Yamashiro L., Feng X., Bierschenk D., Gao Y., Blom H., Henriques-Normark B., Nylén S, & Rothfuchs A.G. (2015). BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming. PLoS Pathogens, 11(10), e1005206.

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Monoclonal Antibody Production and Reagents

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MAbs KJ1-26 (anti-DO11.10 clonotype) [30 (link)] and 23G2 (anti-CD45RB) [31 (link)], were prepared from the supernatants of hybridoma cell lines. Purchased Abs were: HRP-goat anti mouse IgG (Southern Biotech), HRP-goat anti mouse IgM (Southern Biotech), and Magnetic anti-goat mouse Ig microbeads, (Miltenyi Biotec). In addition, rabbit gamma globulin (RGG, Pel Freeze Biologicals, Rogers, AR) was purchased. Where indicated, RGG and BSA (Sigma) or whole chicken ovalbumin (OVA) (Sigma) were haptenated using 2,4,6-Trinitrobenzenesulfonic acid (J.T. Baker Chemical), as previously described. Chicken OVA peptide (OVA323-339) was synthesized and supplied by the Wadsworth Center Peptide Synthesis Core Facility. 5- (and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Inc., Eugene, OR) and SEB (Toxin Technology), and ELISPOT kits (Becton Dickenson) were purchased.

Janik D.K, & Lee W.T. (2015). Staphylococcal Enterotoxin B (SEB) Induces Memory CD4 T Cell Anergy in vivo and Impairs Recall Immunity to Unrelated Antigens. Journal of clinical & cellular immunology, 6(4), 1-8.

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