5 6 carboxyfluorescein diacetate succinimidyl ester cfse

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5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) is a fluorescent dye used for cell labeling. It passively diffuses into cells and becomes covalently bound to intracellular proteins, allowing for the tracking of cell division and proliferation.

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CFSE (2779 citations) Carboxyfluorescein diacetate succinimidyl ester (CFSE) (55 citations) Carboxyfluorescein diacetate succinimidyl ester (44 citations) 5-and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (23 citations) 5,6-carboxyfluorescein diacetate succinimidyl ester (9 citations) CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester; (7 citations) 5(6)-Carboxyfluorescein (2 citations)

19 protocols using 5 6 carboxyfluorescein diacetate succinimidyl ester cfse

Lymphocyte Characterization and Cytokine Production

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Lymphocytes were identified by forward and side scatter, and cell identity was assessed using fluorochrome-conjugated antibodies anti-CD3 (clone SK7; eBioscience), anti-CD4⁺ (RPA-T4; BD), anti-CD8 (RPA-T8; BD), anti-CD161 (DX12; BD), and anti-TCR Vα7.2 (3C10, BioLegend). Viable cells were gated using Live/Dead Yellow or Live/Dead Aqua viability dyes (Invitrogen) according to the manufacturer’s instructions. Cells were stained for 20 minutes in the dark at room temperature, washed, and fixed in PBS containing 2% formaldehyde. All samples were acquired on LSRII or LSRFortessa flow cytometers (BD). Cell division was assessed by labeling PBMCs with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) for 10 minutes at 37°C. Staining was quenched by the addition of FBS for 5 minutes on ice. Cells were then washed and cultured as described.
For detection of intracellular cytokines, cells were stimulated with 50ng/mL soluble anti-CD3 (HIT3a; BD) and 3μg/mL soluble anti-CD28 (CD28.2; BD) for 24 hours at 37°C and 5% CO₂, in the presence of brefeldin A (GolgiPlug; BD) for the final 6 hours of stimulation. After stimulation, cells were washed, stained with viability dye and antibodies to surface antigens, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained intracellularly with fluorochrome-conjugated antibody to IFNγ (B27, BD).

Freeman M.L., Morris S.R, & Lederman M.M. (2017). CD161 Expression on Mucosa-Associated Invariant T Cells is Reduced in HIV-Infected Subjects Undergoing Antiretroviral Therapy Who Do Not Recover CD4+ T Cells. Pathogens & Immunity, 2(3), 335-351.

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Fibroblast Activation and TGF-β Signaling

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Propidium iodide (PI) and trypsin were purchased from Sigma (St. Louis, MO, U.S.A.). DMEM, streptomycin and other cell culture supplies were from GIBCOBRL (Grand Island, NY, U.S.A.). Fetal bovine serum was from Hyclone (Logan, UT, U.S.A.). Acridine orange (AO) was obtained from Fluka (Ronkonkoma, NY, U.S.A.). 5(6)-carboxy fluorescein diacetate succinimidyl ester (CFSE) was from Molecular Probes (Eugene, OR, U.S.A.). Recombinant human TGF-β₁, TGF-β₁ and CTGF ELISA kits were purchased from R&D (Minneapolis, MN, U.S.A.). Trizol and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, U.S.A.). SYBR^@Primescript^TM RT-PCR kit was from Takara Biotechnology, Japan. CTGF and TGF-β₁ primary antibody, as well as second antibody goat anti-mouse IgG and Rhodamine (TRITC)-conjugated affinipure goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Lv L., Liu F.R., Na D., Xu H.M., Wang Z.N, & Jiang C.G. (2020). Transforming growth factor-β1 induces connective tissue growth factor expression and promotes peritoneal metastasis of gastric cancer. Bioscience Reports, 40(9), BSR20201501.

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Investigating MenSCs' Immunomodulatory Effects

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To assess the effects of MenSCs on CD4 + T cells proliferation, CD4 + T cells were co-cultured in different settings as mentioned above. Before co-culturing, CD4 + T cells were washed and labeled with 5 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, USA).
To investigate the effects of MenSCs secretome on CD4 + T cell proliferation, a transwell system (Corning, USA) was utilized. Briefly, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4 + T cells were added into the upper chamber and plates were incubated for five days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/mL anti-IL-6 antibody (R&D systems, USA), 10 µg/mL anti-IL10 antibody (R&D systems, USA) or 10 µg/mL anti-TGF-β (R&D systems, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4 + T cells and frequency of Tregs were assessed by flow cytometry.

Aleahmad M., Bozorgmehr M., Nikoo S., Ghanavatinejad A., Shokri M.R., Montazeri S., Shokri F, & Zarnani A.H. (2021). Endometrial mesenchymal stem/stromal cells: The Enigma to code messages for generation of functionally active regulatory T cells. Stem Cell Research & Therapy, 12, 536.

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Evaluating MenSCs Effect on NK Cells

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The effect of MenSCs on NK cell proliferation was assessed in three different settings. In the first setting, purified NK cells were co-cultured in the absence or presence of optimally mitomycin C (Sigma)-inactivated, 25 µg/mL for 1 hrs, MenSCs (25 × 10³ MenSCs/well) at different ratios of MenSCs:NK (1:2-1:128) in a 96-well culture plate by using direct co-culture system for 5 days. NK cells were also co-cultured at the above ratios with MenSCs using the transwell 24-well plate (Corning, USA). In the second setting, MenSCs were pretreated with IFN-γ (25 ng/mL) and/or IL-1β (10 ng/mL) (all from PeproTech EC, UK) for 48 hrs and co-cultured with MenSCs with or without mitomycin C inactivation. In the third setting and to evaluate possible involvement of IL-6, IL-10, and TGF-β in MenSCs-modulated NK cell proliferation, IFN-γ/ IL-1β-pretreated MenSCs were co-cultured with NK cells in both presence and absence of IL-6, IL-10, and TGF-β neutralizing antibodies (10 µg/mL) (all from R&D systems). In all settings, NK cells were labeled with 5 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, USA) before co-culture with MenSCs. Cultures were continued for 5 days, after which NK cells were harvested, stained with propidium iodide (PI) (Sigma), and proliferation was assessed by flow cytometry.

Shokri M.R., Bozorgmehr M., Ghanavatinejad A., Falak R., Aleahmad M., Kazemnejad S., Shokri F, & Zarnani A.H. (2019). Human menstrual blood-derived stromal/stem cells modulate functional features of natural killer cells. Scientific Reports, 9, 10007.

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Tracking Lymphocyte Trafficking Kinetics

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Spleens harvested from age-matched C57BL/6J mice were pooled and homogenized through a 70-µm nylon filter (BD Biosciences). Single-cell suspensions were depleted of erythrocytes, resuspended at 25 × 10⁶ cells/mL 0.1% bovine serum albumin in PBS, and incubated for 10 min at 37°C in the presence of 0.8 μM (entry) or 5 μM (exit) 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) prepared as a 5 μM stock solution in dimethyl sulfoxide (DMSO). Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum, 4 mM L-glutamine, and 55 µM of 2-mercaptoethanol. Cells were washed in Hank's buffered saline solution (HBSS) and resuspended at 200 × 10⁶ cells/mL. Each recipient mouse received 40 × 10⁶ CFSE-labeled cells intravenously. LNs were harvested 2 h or 20 h post-injection via flow cytometry described below. To prevent continued entry of labeled lymphocytes in some exit experiments, mice were adoptively transferred CFSE-labeled cells as described above, followed by IP injection of 80 μg anti-mouse CD62L-blocking antibody (BioLegend) after 2 h of equilibration, LN harvest 26 h after transfer of labeled cells, and flow cytometry.

Habenicht L.M., Albershardt T.C., Iritani B.M, & Ruddell A. (2016). Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy. Oncoimmunology, 5(8), e1204505.

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ARPE-19 Cell Culture Methodology

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Human Retinal Pigment Epithelial (ARPE-19) cell lines were obtained from ATCC (Manassas, VA). DMEM/F12 culture medium, fetal bovine serum, penicillin and streptomycin were from Life Technologies (Grand Island, NY). Gentamicin, Metronidazole, 5(6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes), Rhodamine-phalloidin (F-actin), Trypsin (0.25%)/EDTA (0.1%), TRIzol reagent, High-Capacity cDNA Reverse Transcription Kit, TaqMan gene expression assay (primers/probes) and 8-well chamber slides were obtained from Thermo fisher scientific (Waltham, MA). RNeasy kit was from Qiagen (Germantown, MD). The universal power block was obtained from Biogenex Laboratories (Fremont, CA). DAPI (4′,6-Diamidino-2′-phenylindole dihydrochloride) from Sigma-Aldrich Co. LLC (St. Louis, MO).

Arjunan P., Swaminathan R., Yuan J., Al-Shabrawey M., Espinosa-Heidmann D.G., Nussbaum J., Martin P.M, & Cutler C.W. (2020). Invasion of Human Retinal Pigment Epithelial Cells by Porphyromonas gingivalis leading to Vacuolar/Cytosolic localization and Autophagy dysfunction In-Vitro. Scientific Reports, 10, 7468.

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Multicolor Flow Cytometry for Immune Cells

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Cells were stained with the indicated monoclonal antibodies conjugated or not to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or Cy-chrome 5 (Cy); anti-Human Fas Ligand (Fas-L), anti-human Vβ8 (BD Pharmingen) anti-human CD4 (Biocience), and/or dyes: Annexin-V (BD bioscience), Propidium Iodine (Sigma) 3,3`-diethyloxacarbocyanine iodine (DiOC₂ (3)), 5,6 carboxyfluorescein diacetate succinimidyl ester (CFSE), (Molecular Probes, Eugene, OR, USA). Briefly, cells (1 × 10⁶) were incubated with the appropriate antibody or dye for 20 minutes at room temperature in the dark (26 (link)) and washed with PBS. For Flow cytometry analysis, 30,000 events were collected in linear mode for forward scatter and side scatter and log amplification for FL-1, FL-2, and FL-3 using a FACScan cytometer (BD Biosciences). Background values were obtained with isotype controls (BD Pharmingen). Results were analyzed by using Cell Quest software (BD Immunocytometry System).

Duarte A., Montagna D.R., Pastorini M, & Alemán M. (2023). Apoptosis-mediated inhibition of human T-cell acute lymphoblastic leukemia upon treatment with Staphylococus Aureus enterotoxin-superantigen. Frontiers in Immunology, 14, 1176432.

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Mucosal Immune Response Induction

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Dimodan ® MO 90/D (monoolein) was kindly donated by Danisco (Grindsted, Denmark). Dextran (from Leuconostoc spp., 40 kDa), ovalbumin (Grade VII, from chicken egg white) and dibutyl sebacate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quil-A was obtained from Brenntag Biosector (Frederikssund, Denmark), phosphate buffered saline (PBS) tablets were acquired from Oxoid limited (Basingstoke, England) and Eudragit ® L100-55 (EL100-55) was purchased from Evonik (Darmstadt, Germany). 5,6-Carboxyfluorescein diacetate succinimidyl ester (CFSE) and CellTrace TM Violet Cell Proliferation Kit (CTV) were purchased from Molecular Probes ® (Eugene, OR, USA). OVA257-264 peptide (SIINFEKL) was acquired from Mimotopes (Clayton, Australia). PeCy7 anti-CD8, propidium iodide and HRP Goat anti-mouse IgG were from BioLegend ® and APC-H7 anti-CD4, PE anti-Vα2, biotin anti-Vβ5 and APC streptavidin from BD Pharmingen TM . Complete mini protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Mannheim Germany) and Mouse Anti-OVA IgA Antibody Assay Kits from Chondrex inc. (WA, USA). All other chemicals were of analytical grade and used as received. Milli-Q water (Merck Millipore, Darmstadt, Germany) was used throughout the study.

von Halling Laier C., Gibson B., Moreno J.A.S., Rades T., Hook S., Nielsen L.H, & Boisen A. (2019). Microcontainers for protection of oral vaccines, in vitro and in vivo evaluation. Journal of controlled release : official journal of the Controlled Release Society, 294.

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CD8+ T Cell Proliferation Assay

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To measure CD8 T cell proliferation, 96-well round-bottom plates were pre-coated with 100 µl anti-CD3ε (BD Biosciences 145-2C11, 1 µg/ml) and anti-CD28 (BD Biosciences 37.51, 10 µg/ml) and incubated at 4°C for 24h. Splenic CD8 T cells were purified using Dynabeads Untouched Mouse CD8 Cell isolation kit (Invitrogen) according to the manufacturer’s protocol. Purified CD8 T cells were labeled with 1 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) at 37°C for 20 min in serum-free RPMI. Excess dye was removed by washing the labeled cells with RPMI 1640 supplemented with 2 µM glutamine, 100 U/ml penicillin/streptomycin, 10% fetal calf serum and 30 µM 2-mercapatoethanol (complete media). Sorted Gr-MDSC (CD45⁺CD11b⁺ Gr-1^highLyC^int), Mo-MDSC (CD45⁺CD11b⁺Gr-1^intLy6C^high) or TAM (CD45⁺CD11b⁺Gr-1^intLy6C^int) were incubated at titrating numbers with 1 × 10⁵ CFSE-labeled labeled T cells in complete media. After 48h, cell cultures were stained for surface markers with CD8α-e450 (BD Biosciences 53-6.7, 1:200) and Thy1.1 (CD90.1)-PercP (BD Biosciences OX-7, 1:200) in PBS/2.5% FBS, followed by Annexin-V-APC (BD Biosciences) staining according to the manufacturer’s protocol, and immediately analyzed on a FACSCanto II. The percentages of apoptotic (Annexin-V) or proliferating (CFSE-dilution) T cells were determined by gating on CD8⁺ Thy1.1⁺ cells.

Stromnes I.M., Brockenbrough S., Izeradjene K., Carlson M.A., Cuevas C., Simmons R.M., Greenberg P.D, & Hingorani S.R. (2014). Targeted depletion of a MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity. Gut, 63(11), 1769-1781.

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Cytotoxicity Assays for Evaluating NK and ADCC

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Cytotoxicity assays were conducted as described previously (13 (link),18 (link)). In summary, splenocytes were collected from vaccinated mice 18 days after being challenged with B16F10 and depleted of erythrocytes by the lysis buffer (Beyotime Biotechnology, Shanghai, China). The collected splenocytes were resuspended with RPMI 1640 medium with 15% FBS for further use. YAC-1 and B16F10 cells were collected, marked with the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; 20 µg/mL) (Invitrogen, Carlsbad, CA, USA) at 37 ℃ for 30 minutes and then washed with cold PBS. To estimate the cytotoxicity of natural killer (NK) cells, NK effector cells were cultured in Corning U-bottom 96 well plates (3×10⁶ cells per well), then were incubated with YAC-1 target cells (1×10⁵cells per well) for 6 hours at 37 °C in a cell culture incubator. For the antibody-dependent cell-mediated cytotoxicity (ADCC) assay, a total of 1×10⁵ B16F10 target cells were incubated with 3×10⁶ effector splenocytes in a medium containing serum from vaccinated mice at a 30:1 ratio at 37 ℃ for 6 hours. After incubation, the different effector-target cells were harvested and rinsed with pre-chilled PBS 3 times. Finally, all the cell samples were labeled with 7-AAD (Beyotime Biotechnology) at room temperature for 10 minutes and detected using FCM (BD), followed by analysis on FlowJo X (BD).

Shi F., Xue R., Xu H., Mei F., Bao X., Dou J, & Zhao F. (2022). Mucin 1 downregulation decreases the anti-tumor effects of melanoma vaccine. Annals of Translational Medicine, 10(24), 1361.

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