Ifn γ bv510

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Sourced in United States

IFN-γ-BV510 is a fluorochrome-conjugated antibody that binds to interferon gamma (IFN-γ), a cytokine involved in immune response. It can be used for the detection and quantification of IFN-γ-expressing cells in flow cytometry applications.

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IFN-γ (422 citations) Anti IFN‐γ BV510 (3 citations)

7 protocols using ifn γ bv510

Intracellular Staining and Cytokine Detection

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For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).

Lee H., Jeon J.H., Lee Y.J., Kim M.J., Kwon W.H., Chanda D., Thoudam T., Pagire H.S., Pagire S.H., Ahn J.H., Harris R.A., Kim E.S, & Lee I.K. (2022). Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation. Cellular and Molecular Gastroenterology and Hepatology, 15(2), 439-461.

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Characterization of NK Cell Function

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PBMC were isolated from non-cancer healthy donor controls by density gradient centrifugation and seeded at a density of 1 × 10⁶ cells/ml RPMI supplemented with 10% FBS and 1% pen/strep. Cells were treated with ACM or TCM from non-obese or obese OAC patients for 2 or 24 h. Cells were subsequently stimulated with 30 ng/ml of IL-12 and 100 ng/ml of IL-15 (Immunotools) for a total of 18 h. After 14 h of stimulation, CD107a-PE-Cy7 (Biolegend) was added for 1 h, followed by addition of 10 µg/ml of Brefeldin A (Sigma) for the remaining three hours. Surface staining for CD56-FITC-Viobright (Miltenyi Biotech) and CD3-APC-Cy7 (Biolegend) was performed. Subsequent intracellular staining for GranzymeB-PE-Cy5, TNF-α-APC, IL-10-BV421 and IFN-γ-BV510 (Biolegend) was performed using FIX&PERM Cell Fixation and Permeabilization Kit (Nordic MUBio). Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (BD Biosciences).

Mylod E., O’Connell F., Donlon N.E., Davern M., Marion C., Butler C., Reynolds J.V., Lysaght J, & Conroy M.J. (2024). Real-time ex vivo monitoring of NK cell migration toward obesity-associated oesophageal adenocarcinoma following modulation of CX3CR1. Scientific Reports, 14, 4017.

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Multicolor Flow Cytometry for Immune Profiling

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Multi-color flowcytometric analysis was performed on cells according to standard procedures using anti-human mAbs that cross-react with rhesus macaques. The following antibodies were used at predetermined optimal concentrations: CD45 (BD clone D058-1283), CD3 APC-Cy7 (BD clone SP34-2), CD4 BV605 (BD clone L200), CD8 BV650 (BD clone SK1), CD69 PE-CF594 (BD clone FN50), CD161 PE (Biolegend clone HP- 3G10), TCR γδ PE-Cy7 (BD clone B1), TCR Vδ1 FITC (ThermoScientific clone TS8.2), TCR Vδ2 FITC (ThermoScientific clone 15D), TCR Vα7.2 BV421 (Biolegend clone 3C10), and Aqua Live/Dead amine dye-AmCyan from Invitrogen (Waltham, MA). Surface staining was carried out by standard procedures as earlier described (23 (link)). Flow cytometric acquisition was performed on the BD Fortessa instrument driven by the FACS DiVa software for at least 100,000 CD3+ T cells in PBMC or at least 10,000 CD3+ T cells for rectal biopsy lymphocytes. The data acquired were analyzed using FlowJo software (version 10.7.1; TreeStar, Ashland, OR). For evaluation of cytokine production, intracellular cytokine staining with IFN-γ BV510 (Biolegend clone 4S.B3), IL-17 PerCP-Cy5.5 (eBioscience clone eBio64DEC17), IL-22 APC (Invitrogen clone IL22JOP), and TNF-α Alexa Fluor 700 (BD clone MAb11) were utilized.

Walker E.M., Slisarenko N., Gerrets G.L., Grasperge B.F., Mattison J.A., Kissinger P.J., Welsh D.A., Veazey R.S., Jazwinski S.M, & Rout N. (2021). Dysregulation of IL-17/IL-22 Effector Functions in Blood and Gut Mucosal Gamma Delta T Cells Correlates With Increase in Circulating Leaky Gut and Inflammatory Markers During cART-Treated Chronic SIV Infection in Macaques. Frontiers in Immunology, 12, 647398.

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Multicolor Flow Cytometry Panel

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The following anti-mouse antibodies were used: CD3-Pacific Blue, CD4-PEridinin chlorophyll protein (PerCP)-Cy5.5, CD8-allophycocyanin (APC)-Cy7, CD69-phycoerythrin (PE), CD44-fluorescein isothiocyanate (FITC), CD62L-APC, CD103-PE-Cy7, CD69-FITC, IFN-γ-APC, IFN-γ-BV510, IL-17-PE-Cy7, IL-17-BV650, CD11b-APC-Cy7, CD11c-APC, CD80-FITC, CD86-PerCP-Cy5.5, CD40-PE, CD4-PE, CD4-APC, CD4-FITC, CD3-BV510, and CD3-BV650 from Biolegend, USA.
Alexa Fluor 647 secondary antibody or isotype control IgG-APC was from Abcam.
p-P38, P38, pSTAT3, pSTAT4, and β-tubulin were obtained from Cell Signaling Technology.
I-A(b)_4-17ESAT-6 MHC-II tetramer to analyze the M. tuberculosis-specific CD4⁺ T cell responses was procured from NIH Tetramer Facility, USA (USA).

Kumari A., Pahuja I., Negi K., Ghoshal A., Mukopadhyay S., Agarwal M., Mathew B., Maras J.S., Chaturvedi S., Bhaskar A, & Dwivedi V.P. (2023). Withaferin A Protects against Primary and Recurrent Tuberculosis by Modulating Mycobacterium-Specific Host Immune Responses. Microbiology Spectrum, 11(2), e00583-23.

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Multiparametric Flow Cytometry of Immune Cells

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Following antibodies were used for this study:
Anti-Mouse: CD3-Pacific Blue, CD4-PErCPCy5.5, CD8-APCCy7, CD69-PE, CD44-FITC, CD62L-APC, CD103-PeCy7, CD69-FITC, IFNγ-APC, IFNγ-BV510, IL17-PECy7, IL17-BV650, CD11b-APCCy7, CD11c-APC, CD80-FITC, CD86-PerCPCy5.5, CD40-PE, CD4-PE, CD4-APC and CD4-FITC, CD3-BV510, CD3-BV650 from Biolegend, USA.
Anti-human: CD3-PE, CD4-APCCy7, CD8-Pacific Blue, CD45RO (PerCPCY5.5), CCR7 (PeCy7), CD69 (Alexa Fluor 700) from BD Biosciences.
Anti-human/anti-mouse: STAT3, p-STAT3, STAT4, p-STAT4, AKT, p-AKT, FOXO1, p-FOXO1, NFκB, p-NFκB, BLIMP1 and β-Tubulin from Cell Signaling Technology.

Pahuja I., Negi K., Kumari A., Agarwal M., Mukhopadhyay S., Mathew B., Chaturvedi S., Maras J.S., Bhaskar A, & Dwivedi V.P. (2023). Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis. PLOS Pathogens, 19(3), e1011165.

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Multiparametric Immune Profiling of Splenocytes

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Splenocytes were incubated with either 20 ng mL⁻¹ phorbol myristate acetate (Sigma‐Aldrich) and 1 ug mL⁻¹ ionomycin (Cell Signaling Technology), S protein‐derived peptides (GenScript), N protein‐derived peptides (GenScript), or control (no stimulation) in 1X Brefeldin A and 1X Monensin (Biolegend) solutions for 6 h at 37 °C. Next, the cells were washed with PBS and incubated for 30 min with Fixable Viability Dye eFluor 780 (eBioscience) in PBS (1:1000 dilution) at 4 °C. Cells were then washed once with PBS and twice with PBA before being stained overnight with fluorochrome‐conjugated antibodies (CD3‐FITC (Clone 145‐2C11), CD4‐PerCP‐Cy5.5 (Clone GK1.5), CD8‐AF700 (Clone 53–6.7), CD44‐BV605 (Clone IM7), Biolegend) in PBA at 4 °C. Cells were subsequently washed 3 times with PBA, fixed in 4% PFA, and then permeabilized using a Cyto‐Fast Fix/Perm Buffer Set (Biolegend) according to the manufacturer's protocol. Intracellular staining was performed by incubating the cells with fluorochrome‐conjugated antibodies (IL‐13‐PE (Clone: W17010B), IL‐4‐PE‐Cy7 (Clone: 11B11), IL‐17‐APC (Clone: TC11‐18H10.1), IL‐5‐BV421 (Clone: TRFK5), IFNγ‐BV510 (Clone XMG1.2), Biolegend) in permeabilization buffer for 30 min at 4 °C. Lastly, cells were washed 3 times with permeabilization buffer, resuspended in PBA, and analyzed using the Attune NxT flow cytometer (Thermo Fisher Scientific).

Colombani T., Eggermont L.J., Rogers Z.J., McKay L.G., Avena L.E., Johnson R.I., Storm N., Griffiths A, & Bencherif S.A. (2021). Biomaterials and Oxygen Join Forces to Shape the Immune Response and Boost COVID‐19 Vaccines. Advanced Science, 8(18), 2100316.

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T Cell Cytokine Induction Assay

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Cryopreserved PBMC were thawed, washed, resuspended in RPMI + 10% FBS (R10), and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and incubated for 2 hrs with our panel of 10 LRA. Following this 2-hour incubation period, GolgiPlug (BD, cat. no. 555029) and GolgiStop (BD, cat. no. 554724) were added to each condition at a concentration of 1:1000 and 6:10000 respectively, then incubated at 37°C for an additional 16 hrs. Cells were harvested, pelleted, resuspended, stained for 20 minutes at 4°C with surface staining mAb (CD3-BV605, CD4-BV421, CD8-PerCp-Cy5.5 and LIVE/DEAD Near-IR stain), fixed and permeabilized as described above, and then stained for 30 minutes at 4°C with the intracellular mAb TNF-α- PE/Dazzle-594 (Biolegend cat. no. 502946), IL-2- PE/Cy7 (BD cat. no. 560707) and IFN-γ- BV510 (Biolegend cat. no. 502544). PBMC were washed and fixed in (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis, as described above.

Lilie T., Bouzy J., Asundi A., Taylor J., Roche S., Olson A., Coxen K., Corry H., Jordan H., Clayton K., Lin N, & Tsibris A. (2023). Opioid use does not limit potent low-dose HIV-1 latency reversal agent boosting. medRxiv.

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