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Ifn γ b27

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IFN-γ (B27) is a laboratory reagent that is used to detect and measure the presence of the cytokine interferon-gamma (IFN-γ) in biological samples. IFN-γ is a signaling protein that plays a crucial role in immune system function and response. The IFN-γ (B27) product is designed to facilitate the analysis and quantification of IFN-γ levels in research applications.

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Lab products found in correlation

Brefeldin a, by Merck Group (5 mentions) Ionomycin, by Merck Group (3 mentions) 15 mer peptides, by AnaSpec (2 mentions) Anti cd49d co stimulatory mab, by BD (2 mentions) Tnf mab11, by Thermo Fisher Scientific (2 mentions) Cd69 fn50, by BD (2 mentions) Anti cd28, by BD (2 mentions) Cd4 rpa t4, by BD (2 mentions) Cd3 sk7, by BD (2 mentions) Il 2 mq1 17h12, by BioLegend (2 mentions) Cxcr5 rf8b2, by BD (2 mentions) Cd25 m a251, by BD (2 mentions) Cd8 sk1, by BD (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions) Cd45ro uchl1, by Beckman Coulter (2 mentions) Il 4 8d4 8, by BD (2 mentions) Il 5 jes1 39d10, by BD (2 mentions) Il 13 jes10 5a2, by BD (2 mentions) Cytofix cytoperm, by BD (2 mentions) Foxp3 236a e7, by Thermo Fisher Scientific (2 mentions)

9 protocols using ifn γ b27

1

Peptide-pulsed CD4+ T Cell Assay

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Peptide-pulsed autologous CD4+ T cells were used as antigen presenting cells (APC) for stimulation assays. APC were labeled with 5μM CellTrace Violet (CTV) as recommended by the manufacturer (Life Technologies, Carlsbad, CA) and pulsed with the Gag peptide for 30 m at room temperature and washed D-PBS and then resuspended in T-cell medium. 1 × 106 CD4+ T cells or T-cell clones were co-cultured with an equal amount of pulsed or unpulsed APC for 6 h with monensin (Golgi stop; BD Biosciences) and anti-human CD107a (H4A3; BD Biosciences). Cells were surface stained, fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and stained for IFN-γ (#B27) and MIP1-β (# D21-1351) both from BD Biosciences.
Ayala V.I., Trivett M.T., Coren L.V., Jain S., Bohn P.S., Wiseman R.W., O’Connor D.H., Ohlen C, & Ott D.E. (2016). A Novel SIV Gag-Specific CD4+ T-Cell Clone Suppresses SIVmac239 Replication in CD4+ T Cells Revealing the Interplay between Antiviral Effector Cells and Their Infected Targets. Virology, 493, 100-112.
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2

Quantification of SIV-specific CD8+ T Cells

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SIV-specific CD8+ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail45 (link). Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3+, CD4−, CD8+ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.
Fukazawa Y., Lum R., Okoye A.A., Park H., Matsuda K., Bae J.Y., Hagen S.I., Shoemaker R., Deleage C., Lucero C., Morcock D., Swanson T., Legasse A.W., Axthelm M.K., Hesselgesser J., Geleziunas R., Hirsch V.M., Edlefsen P.T., Piatak M J.r., Estes J.D., Lifson J.D, & Picker L.J. (2015). A B cell follicle sanctuary permits persistent productive SIV infection in elite controllers. Nature medicine, 21(2), 132-139.
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3

Quantification of SIV-specific CD8+ T Cells

Cited 1 time
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SIV-specific CD8+ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail45 (link). Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3+, CD4−, CD8+ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.
Fukazawa Y., Lum R., Okoye A.A., Park H., Matsuda K., Bae J.Y., Hagen S.I., Shoemaker R., Deleage C., Lucero C., Morcock D., Swanson T., Legasse A.W., Axthelm M.K., Hesselgesser J., Geleziunas R., Hirsch V.M., Edlefsen P.T., Piatak M J.r., Estes J.D., Lifson J.D, & Picker L.J. (2015). A B cell follicle sanctuary permits persistent productive SIV infection in elite controllers. Nature medicine, 21(2), 132-139.
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4

NK Cell Immunophenotyping Protocol

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NK cells were stained with antibodies recognizing CD56 (NCAM16.2), CD3 (UCHT1), CD34 (581), CD4 (RPA-T4), CD19 (HIB19), TNF-α (Mab 11), and IFN-γ (B27) from BD Biosciences (Franklin Lakes, NJ, USA), CD107a (H4A3), CD34 (561), and CXCR4 (12G5) from BioLegend (San Diego, CA, USA), along with propidium iodide (BD Biosciences), annexin V (BioLegend), and/or a Live/Dead fixable aqua dead cell stain kit (Thermo Fisher Scientific). Cells were examined on a BD Biosciences LSRFortessa instrument, and data were analyzed using FlowJo v10.1 (BD Biosciences). Statistical analyses were performed with GraphPad Prism 8.4.1.
Allan D.S., Chakraborty M., Waller G.C., Hochman M.J., Poolcharoen A., Reger R.N, & Childs R.W. (2021). Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion. Molecular Therapy. Methods & Clinical Development, 20, 559-571.
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5

Activation and Phenotyping of T Cells

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Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 106 cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3+ CD4+ CD45RO+ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described 33 (link).
Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.
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6

Multiparametric Analysis of T Cell Subsets

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We stained human PBMCs with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), CD25 (M-A251; BD Biosciences), CD45RA (ALB11; Beckman Coulter), CCR7 (MAB197; R&D Systems), Helios (22F6; BioLegend), Foxp3 (236A/E7; eBioscience), IL4 (MP4-25D2; BD Biosciences), IL-17 (eBio64DEC17; eBioscience), IFN-γ (B27; BD Biosciences) and a Fixable Viability Dye eFluor780 (eBioscience). The cytokine-producing capacity of T cell subsets was assessed after PBMCs were stimulated (at a density of 5 × 105 cells per 100 μL) for 5 h with 50 ng/ml PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich) and 2 mg/ml of ionomycin (Sigma-Aldrich) in the presence of 10 mg/ml of brefeldin A (Sigma-Aldrich). Cells were fixed and made permeable with the Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer's instructions (eBioscience). For flow cytometric analysis of human intestine, IEL and LPL were stained with monoclonal antibodies to CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), IL-7Rα (HIL-7R-M21; BD Pharmingen), and c-Kit (104D2; BD Biosciences).
Frascoli M., Jeanty C., Fleck S., Moradi P.W., Keating S., Mattis A.N., Tang Q, & MacKenzie T.C. (2016). Heightened immune activation in fetuses with gastroschisis may be blocked by targeting IL-5. Journal of immunology (Baltimore, Md. : 1950), 196(12), 4957-4966.
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7

Detailed Immunophenotyping of T-cell Subsets

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The following antibodies were used: CD80 (L307.4; BD Biosciences), CD86 (FUN-1; BD Biosciences), CD86 (IT2.2, BioLegend), CD25 (BC96; eBioscience), FoxP3 (236A/E7; eBioscience), CTLA-4 (BNI3; BD Biosciences), IFN-γ (B27; BD Biosciences), CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD45RA (HI100; eBioscience), CD38 (HIT2; BD Biosciences) and HLA-DR (G46-6; BD Biosciences). For surface marker staining, cells were washed and re-suspended in 50 µl of FACS buffer (PBS and 2% Goat serum) containing antibodies conjugated with fluorochromes. Reactions were incubated for 30 min on ice. Cycling CTLA-4 was stained for 30 min at 37°C. For intracellular staining of FoxP3, a FoxP3 staining kit (eBioscences) was used according to the manufacturer’s instructions. Flow cytometry data were analysed by FlowJo (TreeStar, Ashland, Oregon, USA).
Soskic B., Jeffery L.E., Kennedy A., Gardner D.H., Hou T.Z., Halliday N., Williams C., Janman D., Rowshanravan B., Hirschfield G.M, & Sansom D.M. (2021). CD80 on Human T Cells Is Associated With FoxP3 Expression and Supports Treg Homeostasis. Frontiers in Immunology, 11, 577655.
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8

Cytokine Profiling of CNS-Derived T Cells

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Freshly purified CNS-MNCs were promptly stimulated with a cocktail of phorbol-12-myristate-13 acetate (PMA) and Ionomycin that included BrefeldinA (Leukocyte Activation Cocktail with BD GolgiPlug, BD Biosciences, San Jose, CA) for 6 hrs (Slywester, 2014 ). After stimulation CNS-MNCs were stained with CD4 (RPA-T4, eBioscience, San Diego, CA), CD8β (2ST8.5H7, Beckman Coulter, Brea, CA) and in some instances CD3 (SP34-2, BD Biosciences). Cells were subsequently fixed and permeabilized using fixation buffer and permeabilization buffer (Biolegend, San Diego, CA). Intracellular cytokine staining was performed using IL-17A (eBio64CAP17, eBioscience) and IFN-γ (B27, BD Biosciences). All flow cytometry data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR).
Blair T.C., Manoharan M., Rawlings-Rhea S.D., Tagge I., Kohama S.G., Woltjer R.L., Pollaro J., Rooney W.D., Sherman L.S., Bourdette D.N, & Wong S.W. (2015). Immunopathology of Japanese macaque encephalomyelitis is similar to multiple sclerosis. Journal of neuroimmunology, 291, 1-10.
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9

Activation and Phenotyping of T Cells

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Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 106 cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3+ CD4+ CD45RO+ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described 33 (link).
Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.
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