Anti cd69 fitc

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Anti-CD69 FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD69 antigen expressed on the surface of activated T cells, B cells, and natural killer cells. It can be used for the detection and quantification of these activated immune cells by flow cytometry.

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7 protocols using anti cd69 fitc

Th1/Th2 Cell Phenotyping

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Cell suspensions from Th1/Th2 cell lines at eight days post-re-stimulation were labeled with optimal concentrations of the following labeled monoclonals: PE-anti-CD4 (GK1.5), PE-anti-CD40L (MR1), PE-anti-ICOS (7E.17G9), PE-Rat-IgG2b isotype control, FITC-anti-CD4 (GK1.5), FITC-anti-CD69 (H1.2 F3), FITC-anti-CD3 (145-2C11), FITC-anti-CD5 (53–7.3), FITC-anti-CD54 (YN1/1.7.4), FITC-anti-CD127 (A7R34), FITC-Rat IgG2b isotype control; all from eBioscience; FITC-anti-OX40 (OX-86) from Serotec, UK; APC-anti-CD62L (MEL-14) (ImmunoTools, Friesoythe, Germany); and V500-anti-CD4 (RM4-5) (BD Biosciences, USA). Data were collected on FACSCalibur (BD Biosciences, USA) and analyzed with CellQuest software (BD Biosciences, USA) and FlowJo software (Tree Star, Inc., Ashland, Oregon, USA).

Reynolds C., Chong D., Raynsford E., Quigley K., Kelly D., Llewellyn-Hughes J., Altmann D, & Boyton R. (2014). Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile. BMC Biology, 12, 32.

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Multicolor Flow Cytometry Profiling of Immune Cell Subsets

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After Fc receptors blocking (BD Biosciences, USA), an appropriate concentration of the fluorescence-labeled antibody was used for the staining of surface antigens at 4 °C for 30 min in dark place. Fluorochrome-conjugated monoclonal antibodies of cellular markers: PercpCy5.5-anti-CD3, FITC-anti-IL-17A, PE-anti-CD4, PE-Cy7-anti-NK1.1 (BD Bioscience, USA). FITC-anti-CD69, FITC-anti-IFN-γ; PE-anti-FasL, PE-anti-TRAIL, PE-anti-CD107a, and APC-anti-NKG2D (eBioscience, San Diego, CA). The intracellular cytokine staining, including INF-γ and IL-17A, was using Mouse Intracellular Cytokine Staining Starter Kit (BD Biosciences, USA), according to the kit’s instructions. Samples were measured by a BD Accuri C6 plus flow cytometer (BD Biosciences, USA), and the data were managed using BD Accuri C6 plus analysis (BD Biosciences, USA).

Han X., Huang T, & Han J. (2019). Cytokines derived from innate lymphoid cells assist Helicobacter hepaticus to aggravate hepatocellular tumorigenesis in viral transgenic mice. Gut Pathogens, 11, 23.

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Activated and Memory T Cell Analysis

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Ten days after the third immunization, splenocytes were collected from OVA antigen-immunized C57BL/6 mice. Frequencies of activated T cells and memory T cells in splenocytes were measured by flow cytometry. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (4.0×10 6 cells/mL) and re-stimulated with peptide for 60 h. Cells stimulated with MHC I peptide (OVA 257-264 , 1 μg/mL) were stained with the following set of fluorochromeconjugated anti-mouse antibodies: Alexa Fluor 488-anti-CD8α, PE-anti-CD44, APC-anti-CD62L, and eFluor 450anti-CD69 (eBioscience). Cells stimulated with MHC II peptide (OVA 323-339 , 2 μg/mL) were stained with the following set of fluorochrome-conjugated anti-mouse antibodies: eFluor 450-anti-CD4, PE-anti-CD44, APC-anti-CD62L, and FITC-anti-CD69 (eBioscience). After washing, cell samples were examined by Beckman Coulter CyAn ™ ADP flow cytometer, and data were analyzed by Summit software (version 4.3).

Zhang W., Wang L., Yang T., Liu Y., Chen X., Liu Q., Jia J, & Ma G. (2015). Immunopotentiator-Loaded Polymeric Microparticles as Robust Adjuvant to Improve Vaccine Efficacy. Pharmaceutical research, 32(9).

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Multiparametric Flow Cytometry of Immune Cells

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For immunofluorescence staining, dead cells were excluded with the Near-IR Dead-Cell stain (Invitrogen). Antibodies used were: anti-CD3 PE-Cy7 or APC, anti-CD8 PerCP-Cy5.5 or eFluor 450, anti-CD69 FITC (eBioscience); anti-CD161 PE or APC, anti-CD4 VioGreen (Miltenyi Biotec); anti-Vα7.2 PE or FITC or PE-Cy7, anti-CD107α PE-Cy7, anti-Granzyme A PerCP-Cy5.5, anti-Perforin Pacific Blue, anti-granulysin PE, anti-FasL PE (BioLegend); anti-Granzyme B AlexaFluor700, anti-Perforin FITC, anti-Ki67 FITC (BD Biosciences), anti-Granzyme B APC (Invitrogen); anti-Granzyme K FITC (Immunotools); anti-T-bet PE (Santa Cruz Biotechnology); anti-Granzyme A FITC, anti-Blimp1 AlexaFluor488 (R&D); anti-CD8β PE (Beckman Coulter).
Data were collected on the flow cytometers LSRII (BD Biosciences) or MACSQuant (Miltenyi Biotec), and was analyzed using FlowJo v9.6 (TreeStar). For ImageStream analysis, see Supplementary Methods.

Kurioka A., Ussher J.E., Cosgrove C., Clough C., Fergusson J.R., Smith K., Kang Y.H., Walker L.J., Hansen T.H., Willberg C.B, & Klenerman P. (2014). MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets. Mucosal Immunology, 8(2), 429-440.

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Isolate and Stimulate Murine Naïve CD4+ T Cells

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Spleen and lymph nodes were gently mechanically disrupted through a 70 μm filter, lysed for red blood cells with ACK Lysing buffer (Lonza) and stained with the following Abs (all obtained from ebiosciences): anti-NK-1.1-FITC (clone PK136), anti-CD45R-FITC (clone RA3-6B2), anti-CD11c-FITC (clone N418), anti-CD11b-FITC (clone M1/70), anti-CD69-FITC (clone H1.2F3), anti-CD4-PerCP-Cy 5.5 (clone RM4-5), anti-CD90.2-APC (clone 53-2.1), anti-CD62L-APC-Alexa 780 (clone MEL-14), anti-CD44-FITC (clone IM7), anti-Gr1-FITC (clone RB6-8C5) and anti-CD25-FITC (clone PCS1). Cells were double FACS-sorted through a CD90.2+ CD4+ CD62L+ CD25− CD69− CD44− CD11c− CD11b− NK1.1-CD45R-Gr1- gate using a Synergy 3200 BSC machine (Sony Biotechnology) into complete culture medium (CM: RPMI 1640, 1% Penicillin/Streptomycin (Life technologies), 10% FBS, 28.6 μM β-2-mercaptoethanol (Sigma). A human naïve CD4+ T cell isolation kit II (Miltenyi) were used to isolate CD4⁺ T cells from the peripheral blood of healthy human volunteers. CD4⁺ T cells were co-incubated with 10 μg/mL unconjugated, biotinylated or rhodmaine-conjugated Pam₃CSK₄ (Invivogen) at 37° C for 3 hrs in CM, centrifuged at 250 × g, and washed three times with 15 mls CM prior to an experiment. Plasma membrane staining was conducted with Cell Mask Deep Red (Thermo Fisher Scientific) in accordance with manufacturers recommendations.

Ibrahim M., Scozzi D., Toth K.A., Ponti D., Kreisel D., Menna C., De Falco E., D’Andrilli A., Rendina E.A., Calogero A., Krupnick A.S, & Gelman A.E. (2017). Naïve CD4+ T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion. Journal of immunology (Baltimore, Md. : 1950), 200(2), 847-856.

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Multi-parameter Flow Cytometry for Immune Profiling

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For flow cytometry analysis anti-CD45 AF700, anti-CD45.2 APC-eF780, anti-CD45.1 eF480, anti-CD90.2 APC, anti-CD69 FITC, anti-CD90.1 PE-Cy7, anti-CCR7 PE (eBioscience), anti-CD4 PE TexasRed (Invitrogen), anti-CD8b PerCp/Cy5.5, and anti–IFN-γ PE-Cy7 (BioLegend) were used. For in vivo CD4 depletion mice received i.p. injections of 250 µg GK1.5 on days −5 and −2 before challenge. For in vivo CXCR3 blockade mice received i.p. injections of 250 µg anit-CXCR3-173 mAb (BioXcell) one day before challenge. For intravascular staining mice were injected with 3 µg FITC anti-CD45.2 (eBioscience) 3 min before they were euthanized by CO₂. The data from flow cytometry were collected using LSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star).

Glennie N.D., Yeramilli V.A., Beiting D.P., Volk S.W., Weaver C.T, & Scott P. (2015). Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection. The Journal of Experimental Medicine, 212(9), 1405-1414.

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CD69 Expression in Activated PBMCs

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PBMCs were cultured and treated as previously described. After 24 h, cells were harvested and stained for the early T cell activation marker CD69 (anti-CD69-FITC) as well as anti-CD3-PerCP antibodies (eBioscience, San Diego, CA, USA) and analyzed by flow cytometry (FacsVerse, BD Bioscience, USA) using FlowJo 10.0.7 software. Ten thousand events were recorded per sample. The experiment was performed using PBMCs isolated from four healthy donors.

Alencar-Silva T., de Barcelos S.M., Silva-Carvalho A., Sousa M.G., Rezende T.M., Pogue R., Saldanha-Araújo F., Franco O.L., Boroni M., Zonari A, & Carvalho J.L. (2024). Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro. Cells, 13(10), 813.

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