Anti cd25 fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD25-FITC is a fluorescein isothiocyanate (FITC) conjugated antibody that binds to the CD25 antigen expressed on activated T cells, regulatory T cells, and certain other cell types. It is used for the identification and quantification of these cell populations in flow cytometry applications.

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CD25-FITC (11 citations) FITC-conjugated rat anti-mouse CD25 (2 citations) FITC-conjugated anti-CD25 antibody (2 citations) FITC-conjugated anti-canine CD25 mAb (2 citations) FITC-anti-CD25 antibody (2 citations) Anti-CD4-FITC/CD25-APC cocktail (2 citations)

8 protocols using anti cd25 fitc

Flow Cytometry, ELISA, and Western Blot Analysis

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For flow cytometry, cells were washed in PBS supplemented with 1% FCS, 1% Sodium Azide and 3 mM EDTA (FACS buffer) and stained with a cocktail of anti-CD3 PE, anti-CD4 APC-Cy7 and anti-CD25 FITC (eBioscience). Cells were then fixed and permeabilized using eBioscience FoxP3 staining kit (77-5775-40) and stained with anti-Foxp3 APC. Samples were examined using a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Treestar). For ELISA, supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL10 (DY417) and IFNγ (DY485) according to the manufacturer's instructions (R&D systems). For Western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Triton-x, 1 mM EDTA) using mechanical homogenisation and sonication. Western blotting was performed on tissue suspensions as we have described previously [30] (link). Primary antibodies used were: anti-MOG (Millipore, MAB5680), anti-myelin basic protein (MBP) (Abcam ab40390), anti-actin (ECM Biosciences, AM2021), anti-neurofilament H (NFH) (1/1000 dilution: Millipore, MAB5539). Secondary antibodies used were: HRP conjugated mouse (Sigma, A8924), and HRP conjugated rabbit (GE Healthcare, NA934).

Pritchard A.J., Mir A.K, & Dev K.K. (2014). Fingolimod Attenuates Splenocyte-Induced Demyelination in Cerebellar Slice Cultures. PLoS ONE, 9(6), e99444.

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Multiparametric Flow Cytometry Analysis

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Liver mononuclear cells (MNCs) were purified by a 40%/70% percoll gradient^{48 (link)} and stained with anti-CD3-PE, anti-CD4-PerCP/Cy5.5, anti-CD8a-APC, or anti-CD25-FITC (eBiosciences) for 30 min at 4 °C in staining buffer (PBS, 3% FCS). For detection of intracellular proteins, cells were first stimulated with PMA (Sigma) and inomycin (Sigma) for 4 h and stopped by addition of Brefeldin A (Sigma). After cell surface staining, cells were fixed and permeabilized (eBiosciences), and then stained respectively with anti-IFNγ-FITC, anti-IL-17 A-PE, or anti-Foxp3-PE. For detection of surface expression of IL-17RA in cloned MSCs, cells were stained with anti-IL-17RA-PE (eBiosciences). All samples were analyzed by flow cytometry on a FACS Calibur flow cytometer (Becton Dickinson).

Han X., Yang Q., Lin L., Xu C., Zheng C., Chen X., Han Y., Li M., Cao W., Cao K., Chen Q., Xu G., Zhang Y., Zhang J., Schneider R.J., Qian Y., Wang Y., Brewer G, & Shi Y. (2014). Interleukin-17 enhances immunosuppression by mesenchymal stem cells. Cell Death and Differentiation, 21(11), 1758-1768.

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Intracellular Cytokine Staining of CD4+ T Cells

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The pre‐treated CD4⁺ T cells were washed, and the following mouse antibodies were added for intracellular staining: anti‐CD4‐PE, anti‐IL‐17A‐FITC, anti‐CD25‐FITC and anti‐Foxp3‐APC (eBioscience, San Diego, CA, USA). The cells were fixed in the 4% paraformaldehyde and were permeabilized in the 1% Triton X‐100 at 25°C. The flow cytometry was performed on a FACS Canto™ II (BD, USA). The events were recorded and then analysed using the FlowJo7.6.1 software (Tree Star, Palo Alto, CA, USA).

Yuan X., Dou Y., Wu X., Wei Z, & Dai Y. (2017). Tetrandrine, an agonist of aryl hydrocarbon receptor, reciprocally modulates the activities of STAT3 and STAT5 to suppress Th17 cell differentiation. Journal of Cellular and Molecular Medicine, 21(9), 2172-2183.

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Multicolor Flow Cytometry of Dendritic Cells

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DCs were harvest after 24 h treatment and stained with different antibodies raised against specific surface markers: Anti-CD11C- PerCP-Cy5.5, Anti-CD86-APC, Anti-CD40-FITC, Anti-CD80-PE-Cy7, Anti-MHCII-PE, Anti-CD274 (PD-L1)-PE, Anti-CD273(PD-L2)-FITC, Anti-OX40-L-APC for 30 mins. Co-cultured cells were harvest and then mixed with different antibodies raised against specific surface markers: Anti-CD4-PerCP-Cy5.5 (eBioscience, The Netherlands), anti-CD25-FITC (eBioscience, The Netherlands), anti-CD69-PE (eBioscience, The Netherlands), anti-CCR-6-PE (eBioscience, The Netherlands), for 30 minutes. Staining for intracellular markers were performed according manufacturer’s protocol, (eBioscience, Foxp3 staining set, Bio connect, The Netherlands). Antibodies used for intracellular markers where anti-Foxp3-PE-Cy7 (eBioscience, the Netherlands), anti-RORγt-PE (eBioscience, The Netherlands). Matching Isotype controls were used to minimize the influence of nonspecific binding and proper gate setting. All incubations were performed on ice and protected from light. In total a minimum of 50,000 cells were counted and analyzed using FACS Canto II and FACS Diva software (BD Biosciences, The Netherlands).

Xiao L., van’t Land B., Engen P.A., Naqib A., Green S.J., Nato A., Leusink-Muis T., Garssen J., Keshavarzian A., Stahl B, & Folkerts G. (2018). Human milk oligosaccharides protect against the development of autoimmune diabetes in NOD-mice. Scientific Reports, 8, 3829.

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Quantifying NK and Treg Cells

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Assessment of NK and Treg cells was done using both flow cytometry (Cytomics FC500 flow cytometer) and ELISA. Monoclonal antibodies (mab) were purchased from (eBioscience, San Diego, CA), anti-CD3-FITC, anti-CD4-PE and anti-CD25-FITC, or anti-CD56-PE. The frequency of each cell subset was calculated based on the percentage of positive cells in the total lymphocyte gate. For intracellular staining, cells were first labelled with relative marker, fixed, and permeabilized using a fix/perm kit according to the manufacturer’s instructions and then labelled with anti-FOXP3-PE or anti-NKp46-PE mab (PCH101; eBioscience).
NKp46 and FOXP3 were measured also in cell lysate by sandwich ELISA for in vitro quantitative measurement (mybiosource.com/prods/ELISA-Kit/mouse/forkhead-box-P3, NKp46). Following cell culture of mouse spleen cells, repeated freezing and thawing cycles were done to damage the cells and release the inside components of the cell, followed by centrifugation at 2000–3000 RPM for approximately 20 min. The supernatants were collected carefully and centrifuged again when sedimentation occurred during storage.

Aboelnazar S., Ghoneim H., Moaaz M., Bahgat E.T, & Shalaby T. (2021). Low-dose interleukin-2-loaded nanoparticle effect on NK and T-reg cell expression in experimentally induced type 1 diabetes mellitus. Przegla̜d Gastroenterologiczny, 16(1), 67-82.

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Quantifying Treg Induction by TolDCs

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To evaluate the recruitment of Tregs induced by TolDCs treatment, the lung cells and splenocytes from animals were disrupted into individual cells. The cells (10⁶) were stained with anti-CD4-APC and anti-CD25-FITC, or the corresponding isotypes, in accordance with the manufacturer's recommendations (eBioscience). After surface staining, the cells were permeabilized using a Cytofix/Cytoperm kit (eBioscience), and the cells were then stained with anti-Foxp3-PE (eBioscience). Fluorescence was measured using a FACS Fortessa cytometer (Becton Dickinson) equipped with BD FACSDiva 6.1 software (Becton Dickinson).

Aragão-França L.S., Rocha V.C., Cronemberger-Andrade A., Costa F.H., Vasconcelos J.F., Athanazio D.A., Silva D.N., Santos E.S., Meira C.S., Araújo C.F., Cerqueira J.V., Cardillo F., Alcântara-Neves N.M., Soares M.B, & Pontes-de-Carvalho L.C. (2018). Tolerogenic Dendritic Cells Reduce Airway Inflammation in a Model of Dust Mite Triggered Allergic Inflammation. Allergy, Asthma & Immunology Research, 10(4), 406-419.

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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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Analyzing NK and Treg Cells via Flow and ELISA

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Assessment of NK and Treg cells was done using both flow cytometry (Cytomics FC500 flow cytometer) and ELISA. Monoclonal antibodies (mab) were purchased from (eBioscience, San Diego, CA), anti-CD3-FITC, anti-CD4-PE and anti-CD25-FITC or anti-CD56-PE. The frequency of each cell subset was calculated based on the percentage of positive cells in the total lymphocyte gate. For intracellular staining, cells were first labeled with relative marker, fixed, and permeabilized using a fix/perm kit according to the manufacturer’s instructions and then labeled with anti-FOXP3-PE or anti-NKp46-PE mab (PCH101; eBioscience).
NKp46 and Foxp3 were measured also in cell lysate by sandwich ELISA for in vitro quantitative measurement (mybiosource.com/prods/ELISA-Kit/mouse/forkhead-box-P3, NKp46). Following cell culture of mouse spleen Cells, repeated freezing and thawing cycles were done to damage cells and let out the inside components of the cell then Centrifugation at 2000-3000 RPM for approximately 20 minutes was done. The supernatants were collected carefully and centrifuged again when sediments occurred during storage.

Aboelnazar S., Ghoneim H., Shalaby T., Bahgat E, & Moaaz M. (2021). Effect of low dose IL-2 loaded chitosan nanoparticles on natural killer and regulatory T cell expression in experimentally induced autoimmune type 1 diabetes mellitus. Central-European Journal of Immunology, 45(4), 382-392.

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