Anti ifn γ percp cy5

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-IFN-γ-PerCP/Cy5.5 is a fluorescently-labeled antibody that binds to the cytokine interferon-gamma (IFN-γ). It is designed for use in flow cytometry applications to detect and quantify IFN-γ-producing cells.

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IFN-γ (1815 citations) Anti-IFN-γ (147 citations) PerCP-Cy5.5 (62 citations) PerCP-Cy5.5-anti-IFN-γ (3 citations) Anti-Mouse IFN-γ PerCP-Cy5.5 (2 citations) Anti-human IFN-γ-Percp-cy5.5 (2 citations)

8 protocols using anti ifn γ percp cy5

Differentiation and Cytokine Profiling of Human CD4+ T Cells

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Naive and memory human CD4⁺ T cells were cultured on anti-CD3 (clone OKT3, 10 μg/mL; BioXCell) and anti-CD28 (clone 9.3, 2 μg/mL; BioXCell) coated plates with 20 U/mL IL-2 in RPMI 1640 (Lonza), supplemented with 10% heat-inactivated FCS (BioSera), penicillin/streptomycin and 2 mM L-glutamine (both Lonza), 2-mercapto-ethanol (Gibco), HEPES buffer (Lonza), 100 mM sodium pyruvate (Gibco), and and 1% nonessential amino acids (Sigma). Cells were split and media replenished as necessary.
Th1, Th2, and Th17 cells were cultured with autologous irradiated APCs (CD4⁻ cells) at a 1:5 ratio, with 100 U/mL IL-2 and 1 μg/mL anti-CD3 (OKT3) in X-VIVO 15 media (Lonza), supplemented with 5% human AB serum (Sigma), penicillin/streptomycin, and L-glutamine. GSK-3 inhibitors were added as above and cells were split as necessary. To determine cytokine production, on day 7 of T-cell culture, cells were stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 4 h, in the presence of Golgi Stop. Cells were stained with fixable viability dye ef780 (eBioscience) and surface-stained with anti-CD4 Alexa700. Following fixation and permeabilization, intracellular staining for anti-IL-10 ef660, anti-IFNγ PerCPCy5.5, anti-IL-17A PE, and anti-IL-4 FITC (all eBioscience) was carried out.

Hill E.V., Ng T.H., Burton B.R., Oakley C.M., Malik K, & Wraith D.C. (2015). Glycogen synthase kinase-3 controls IL-10 expression in CD4+ effector T-cell subsets through epigenetic modification of the IL-10 promoter. European Journal of Immunology, 45(4), 1103-1115.

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Naïve CD4+ T Cell Polarization

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Naïve CD4⁺ T cells (CD4⁺ L-selectin^hi cells) were purified using AutoMacs Magnetic Bead cell separation technology (Miltenyi Biotech) from total lymph node cells isolated from unprimed mice with purity ranging from 98–99.9%. For in vitro activation, 5×10⁵ naïve CD4⁺ T cells were activated in the presence of plate-bound anti-CD3 (1 µg/ml) plus Th1- (200 U/ml IL-2, 40 U/ml IL-12, 10 µg/ml anti-IL-4) or Th17-(10 ng/ml TGF-β1, 50 ng/ml IL-6, 1 µg/ml anti-IFN-γ, 1 µg/ml anti-IL-4, 1 µg/ml anti-IL-2) promoting conditions. On day four the cultured T cells were collected and the percentage of viable cytokine positive cells assessed via flow cytometry. The cells were stained with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit, for 405 nm excitation (Life Technologies), anti-CD4-APC/Cy7 (clone RM4–5), anti-IFN-γ-PerCP/Cy5.5 (clone XMG1.2), and anti-IL-17-APC (clone eBio17B7) (eBioscience). Viable cells (5×10⁵) were analyzed per individual sample using a BD Canto II cytometer (BD Biosciences), and the data were analyzed using FloJo Version 9.5.2 software (Tree Star, Inc).

Najm F.J., Madhavan M., Zaremba A., Shick E., Karl R.T., Factor D.C., Miller T.E., Nevin Z.S., Kantor C., Sargent A., Quick K.L., Schlatzer D.M., Tang H., Papoian R., Brimacombe K.R., Shen M., Boxer M.B., Jadhav A., Robinson A.P., Podojil J.R., Miller S.D., Miller R.H, & Tesar P.J. (2015). Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo. Nature, 522(7555), 216-220.

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Intracellular Flow Cytometry for T Cell Analysis

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Intracellular flow cytometry analysis was performed as previously described (22 (link)). Briefly, 5 × 10⁶ splenocytes were plated in each well of a 24-well plate and incubated with either CMFO (10 µg/mL) or PPD (10 µg/mL) in the presence of 1 µg/mL anti-CD28/CD49d (eBioscience CA, USA). RPMI1640 medium and cell stimulation cocktail (eBioscience, CA, USA) were used as negative and monitoring controls, respectively. Cells were stained with surface markers, including anti-CD4 PE Cy7, anti-CD8a PE, anti-CD44 APC-eFluor^® 780, anti-CD62L FITC mAbs, and intracellular markers anti-IFN-γPerCP-Cy5.5 and anti-IL-2 APC mAbs (all from eBioscience, CA, USA). The stained cells were analyzed by an LSRII multicolor flow cytometer (BD Biosciences, CA, USA). The absolute number of CMFO-specific CD4⁺ or CD8⁺ IFN-γ positive T_EM (effector memory T cells, CD62L^loCD44^hi) and CD4⁺ or CD8⁺ IL-2 positive T_CM (central memory T cells, CD62L^hiCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA). The results are represented as mean ± SD per group (n = 6).

Tian M., Zhou Z., Tan S., Fan X., Li L, & Ullah N. (2018). Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection. Frontiers in Immunology, 9, 310.

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Naïve CD4+ T Cell Polarization

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IFNγ Production Assay for KIR3DL1+ NK Cells

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IFNγ production by KIR3DL1⁺ NK cells was assessed essentially as previously described (8 ). Briefly, 5×10⁵ PBMC were incubated with 5x10⁵ target cells (221 transfectants) in the presence of 2500 U IL-2 for 14 hours, with GolgiPlug (BD Biosciences) added one hour into the incubation. Cells were then washed and stained with anti-CD56-APC (BD Bioscience), anti-CD3-PECy7 (eBioscience) and anti-NKB1-FITC (anti-KIR3DL1; BD Bioscience) prior to fixation with 2% paraformaldehyde. Following permeablisation with 0.2% saponin, cells were stained with anti-IFNγ-PerCPCy5.5 (eBioscience) and analysed by flow cytometry. Data was analysed with FlowJo software, with the percentage of IFNγ producing KIR3DL1⁺ NK cells (CD56⁺, CD3⁻) normalised to the maximal IFNγ output when incubated with the parental 721.221 cell line (or 221.B0801 in the analysis of HLA-B*08:01 mutants).

Saunders P.M., Vivian J.P., Baschuk N., Beddoe T., Widjaja J., O'Connor G.M., Hitchen C., Pymm P., Andrews D.M., Gras S., McVicar D.W., Rossjohn J, & Brooks A.G. (2014). The interaction of KIR3DL1*001 with HLA class I molecules is dependent upon molecular microarchitecture within the Bw4 epitope. Journal of immunology (Baltimore, Md. : 1950), 194(2), 781-789.

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CD4+ T Cell Phenotyping by Flow Cytometry

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CD4⁺ phenotype was analyzed by flow cytometry as previously described [22 (link)]. Briefly, 300 μl peripheral blood were stained with anti-CD4 APC-Cy7 (Immunostep) and anti-CD25 FITC for 30 minutes at 4°C. Then, erythrocytes were lysed and cells were fixed and permeabilized (FOXP3 transcription factor staining kit, eBioscience) and intracellularly stained with anti-FOXP3 PE, anti-IFNγ PerCP-Cy 5.5 and anti-IL17 APC (all eBiosciences) or isotype controls (eBioscience). Samples were analyzed and 50,000 CD4⁺ lymphocytes were acquired in a FACS Canto II (BD) cytometer. CD4⁺ cells were gated and their intensity of IFNγ and IL-17 intracellular staining (measured as mean fluorescence intensity, MFI) was analyzed with FACS Diva v. 2.6 (BD) by subtracting the MFI in the isotype control tube to that of the specific antibody-stained one. Regulatory T cells (Treg) were identified as CD4⁺CD25^highFOXP3⁺.

Rodríguez-Carrio J., Alperi-López M., López P., Ballina-García F.J, & Suárez A. (2016). Non-Esterified Fatty Acids Profiling in Rheumatoid Arthritis: Associations with Clinical Features and Th1 Response. PLoS ONE, 11(8), e0159573.

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Multiparametric Flow Cytometry of Immune Cells

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Flow cytometry was performed on cells from whole spleens or on tumors after digestion in DNAse/Collagenase for 40 min. at 37° C. Single cell suspensions were achieved by filtration through a 70 μm filter. Live/Dead Aqua Fixable Stain (Life Technologies, cat L34957) was used. Antibodies used: anti-CD4-APC (BioLegend cat 100412), anti-CD4-PE (Biolegend cat 100512), anti-CD8a-APCeFlour780 (eBioscience cat 47-0081-82), anti-IFNγ-PerCPCy5.5 (eBioscience, cat 45-7311-82), anti-IL4-APC (cat 17-7041-81). Debris, dead cells, and doublets were gated out before analysis. For IFN-γ, single cells were cultured at 2×10⁶ cells per mL splenocyte media containing GP33 or GP61 LCMV peptides and Brefeldin A for 6 hours before staining. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences).

Schadler K.L., Crosby E.J., Zhou A.Y., Bhang D.H., Braunstein L., Baek K.H., Crawford D., Crawford A., Angelosanto J., Wherry E.J, & Ryeom S. (2014). Immunosurveillance by anti-angiogenesis: tumor growth arrest by T cell-derived thrombospondin-1. Cancer research, 74(8), 2171-2181.

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Immunization and Analysis of Arthritis in Mice

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Flt3L^−/−, WT and Batf3^−/− mice were immunized as previously described [23 (link)] and were inspected three times a week for signs of arthritis by two independent observers. All mice were sacrificed on day 43 after CIA induction. Blood, LNs and paws were harvested for analysis. Single-cell suspensions were obtained and after erythrocyte lysis (red blood cell lysis buffer, 2 min at RT; Sigma), cells were stained with the indicated fluorochrome-conjugated antibodies for surface markers and intracellular cytokines. LN cells were stained using the following markers: anti-TNF (APC, eBioscience), anti-IL-2 (APC, eBioscience), anti-IL-17 (Alexa 488, eBioscience), anti-IFNγ (PercP Cy5.5, eBioscience), anti-IL10 (PE, eBioscience), anti-GM-CSF (PE, eBioscience), anti-B220 (PerCP, eBioscience), anti-CD19 (Alexa 700, eBioscience), anti-GL7 (biotin, eBioscience), anti-IgD (PE, BD Pharmingen), anti-CD38 (FITC, eBioscience), anti-CD95 (APC, eBioscience) and streptavidin (PE-Cy7, eBioscience). Synovial cells were isolated and stained using antibodies against CD11c (PE-Cy7, eBioscience), MHCII (APC-Cy7, eBioscience), CD11b (Alexa 700) and CD103 (FITC, eBioscience). Serum levels of antibodies against chicken collagen type II (cCII) were measured by ELISA. Further details are described in supplementary methods.

Ramos M.I., Garcia S., Helder B., Aarrass S., Reedquist K.A., Jacobsen S.E., Tak P.P, & Lebre M.C. (2020). cDC1 are required for the initiation of collagen-induced arthritis. Journal of Translational Autoimmunity, 3, 100066.

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