Ifnγ bv421

Manufactured by BioLegend

Sourced in United States

IFNγ-BV421 is a fluorochrome-conjugated antibody that binds to the interferon gamma (IFNγ) protein. It can be used for the detection and analysis of IFNγ-expressing cells in flow cytometry applications.

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9 protocols using ifnγ bv421

Immunophenotyping of Lymphocytes

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Cell staining, flow cytometry and intracellular staining was performed as previously described. [26 (link)] Isolated spleen and liver infiltrating lymphocytes were stained with Live Dead Near-IR Dead Cell kit (Invitrogen), CD3-BV785 (Biolegend), CD45-BV510 (Biolegend), CD8-PE-Cy7 (Biolegend), CD4-PE-CF594 (Becton Dickinson), and CD25-PE-Cy7 (Becton Dickinson). Intracellular staining was performed using anti-mouse forkhead box P3 (FoxP3)-AF488 (MF23; BD Pharmingen). Intracellular cell staining for IFNγ and flow cytometry was performed as previously described using IFNγ-BV421 (Biolegend) [26 (link)].

Soares K.C., Rucki A.A., Kim V., Foley K., Solt S., Wolfgang C.L., Jaffee E.M, & Zheng L. (2015). TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner. Oncotarget, 6(40), 43005-43015.

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Multiparametric Flow Cytometry of Immune Cells

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Cells were incubated with Fc Block (1 µg/10⁶ cells; 2.4G2; BD Biosciences) for 15 min. Extracellular staining was performed for 30 min on ice using: CD4‐BV521 (RM4‐5; BioLegend, San Diego, CA, USA), CD45‐BV510 (30‐F11; BioLegend), CD3‐APC (17.A2, BioLegend), CD25‐PE‐Cy7 (PC61; BioLegend), CD8‐PerCPCy5.5 (53‐6.7; BioLegend), B220 (RA3‐6B2; BD Biosciences), CD11b‐PE‐Cy7 (M1/70; BioLegend), Ly6C‐PE (HK1.4; BioLegend), Gr1‐APC‐Cy7 (RB6‐8C5, BioLegend), IA/IE‐BV421 (M5/114.15.2, BioLegend), F4/80‐FITC (BM8, BioLegend), CD11c‐PerCPCy5.5 (N418, BioLegend).
After staining for extracellular proteins, cells were fixed in 4% PFA and permeabilised using 0.1% saponin buffer containing 0.1% bovine serum albumin. Intracellular cytokines were detected with IFNγ‐BV421 (XMG 1.2, BioLegend), IL‐10‐PE (54902, BD Bioscience), and IL‐17A‐AF647 (TC11‐18H10, BioLegend).
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analysed using FlowJo software version 10.6 (Treestar Inc., Ashland, OR, USA).

Denny L., Al Abadey A., Robichon K., Templeton N., Prisinzano T.E., Kivell B.M, & La Flamme A.C. (2021). Nalfurafine reduces neuroinflammation and drives remyelination in models of CNS demyelinating disease. Clinical & Translational Immunology, 10(1), e1234.

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Isolation and Analysis of Tumor-Associated Immune Cells

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At day 21 post tumor implantation, mice were sacrificed using a lethal dose of ketamine/xylazine cocktail. The brain and cervical lymph nodes (LN) were harvested and passed through a 40-μm strainer. A 30–37–60% Percoll gradient (GE Healthcare, Buck-inghamshire, UK) was used to isolate immune cell populations from brain tumors and the draining lymph nodes (LNs). After centrifugation, the 37–60% interface contained lymphocytes, monocytes and microglia in the case of brain tumors, and contained lymphocytes and monocytes in the case of draining LNs.
For flow cytometric analysis, lymphocytes were stained with CD8 PerCp-Cy5.5 Clone: 53–6.7 (eBioscience), CD3 FITC Clone: 17A2 (eBioscience), CD4 APCH7 Clone: GK1.5 (BD Biosciences), FoxP3 PE Clone: NNRF-30 (eBioscience), IFNγ BV421 Clone: XMG 1.2 (Biolegend), LAG-3 APC Clone: C9B7W, PD-1 PE-Cy7 Clone: J43 and fixable aqua L/D stain (Life Technologies). Appropriate isotype controls were used.
All flow cytometry experiments were performed on a LSRII (BD Biosciences) and analysis was performed using FlowJo software (TreeStar, Ashland, OR).

Harris-Bookman S., Mathios D., Martin A.M., Xia Y., Kim E., Xu H., Belcaid Z., Polanczyk M., Barberi T., Theodros D., Kim J., Taube J.M., Burger P.C., Selby M., Taitt C., Korman A., Ye X., Drake C.G., Brem H., Pardoll D.M, & Lim M. (2018). Expression of LAG-3 and efficacy of combination treatment with anti-LAG-3 and anti-PD-1 monoclonal antibodies in glioblastoma. International journal of cancer, 143(12), 3201-3208.

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Isolation and Analysis of Splenic Dendritic Cells

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Spleen mononuclear cells were prepared as previously described [69 (link)]. For studies of cDCs, spleens were treated with deoxyribonuclease I (0.5mg/ml; Worthington Biochemical) and collagenase type 4 (1mg/ml; Worthington Biochemical) for 25 minutes at room temperature, in order to ensure maximal recovery of splenic cDCs. Fluorescently conjugated monoclonal antibodies, anti-mouse B220-Alexa Fluor 700 (RA3-6B2), B220-Pacific blue (RA3-6B2), CD19-FiTC (6D5), CD138-BV605 (281–2), IgD-APCCy7 (11-26c.2a), IgM-PECy7 (RMM-1), TCRβ-Alexa Fluor 700 (H57-597), TCRβ-APC/Cy7 (H57-597), CD4-BV605 (RM4-5), IFNγ-BV421 (XMG1.2), ICOS-PE (7E.17G9), Streptavidin-PE/Cy7, CD45.1-FiTC (A20), CD45.2-Alexa Fluor 700 (104), CD11c-APC (N418), MHCII-Pacific blue (M5/114.15.2), CD8α-PE/Cy7 (53–6.7), CD40-PE (1C10), CD80-PE (16-10A1), CD86-PE (GL1), ICOSL-PE (B7-RP1), PDL1-PE (MIH5), PDL2-PE (TY25), CD49d-Biotin (R1-2), CD11a-FiTC (M17/4), Ki-67-PE (16A8) and Zombie Aqua fixable viability dye were purchased from Biolegend (San Diego, CA). Anti-mouse CD95/Fas-BV421 (Jo2), CXCR5-biotin (2G8), and Bcl6-PerCP/Cy5.5 (K112-91) were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-mouse T-bet-APC (eBio4B10), GL-7-APC (GL-7) and PD1-APC/Cy7 (J43) were purchased from eBioscience. Cell surface and intracellular IFNγ, T-bet and Bcl6 staining was performed as previously described [69 (link),70 (link)].

Sebina I., James K.R., Soon M.S., Fogg L.G., Best S.E., de Labastida Rivera F., Montes de Oca M., Amante F.H., Thomas B.S., Beattie L., Souza-Fonseca-Guimaraes F., Smyth M.J., Hertzog P.J., Hill G.R., Hutloff A., Engwerda C.R, & Haque A. (2016). IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection. PLoS Pathogens, 12(11), e1005999.

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Enrichment and Stimulation of CD8+ T Cells

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Isolated liver infiltrating lymphocytes and splenocytes were enriched for CD8 cells using CD8 negative isolation kits (Life Technologies, Frederick, MD, USA) according to manufacturer's protocol. CD3:CD28 stimulation beads (Life Technologies, Frederick, MD, USA) were added to isolated CD8⁺ T cells and incubated for 12 hours at 37°C in 5% CO₂ according manufacturer's protocol. Golgistop (1:1000; BD Biosciences) was added and incubated for 5 hours at 37°C in 5% CO₂. After removing the beads according to manufacturer's protocol and washing the cells twice with flow buffer, cells were stained with CD8, CD3 and live dead Near-IR stain according to the above protocol. The cells were then washed twice, suspended in cytofix/cytoperm buffer (BD Biosciences), incubated at 4°C for 30 minutes and then washed with Permwash (BD Biosciences). IFNγ-BV421 (Biolegend) antibody was added in Permwash and incubated at 4°C for 20 minutes. Flow cytometry assays were completed on an LSR II flow cytometer.

Soares K.C., Rucki A.A., Wu A.A., Olino K., Xiao Q., Chai Y., Wamwea A., Bigelow E., Lutz E., Liu L., Yao S., Anders R.A., Laheru D., Wolfgang C.L., Edil B.H., Schulick R.D., Jaffee E.M, & Zheng L. (2015). PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T cell infiltration into pancreatic tumors. Journal of immunotherapy (Hagerstown, Md. : 1997), 38(1), 1-11.

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Stimulation of Human Immune Cells

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All cell culture supplies were obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA), unless otherwise specified. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). All TLR7/8 compounds were synthesized and characterized as previously reported^{27 (link)}. Frozen human PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH). Fluorophore labelled anti-human monoclonal antibodies (CD3-FITC, CD56-PE/Cy7, CD11c-PE/Cy7, CD19-PE, GranzymeB-PE, IL-6-APC, TNF-α-APC/Cy7, IFNγ-BV421 and IL-2-BV605) and Brefeldin A were purchased from Biolegend (San Diego, CA).

Khanna V., Kim H., Zhang W., Larson P., Shah M., Griffith T.S., Ferguson D, & Panyam J. (2021). Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC). Scientific Reports, 11, 3346.

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PBMC Stimulation and Cytokine Assessment

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Healthy donor PBMCs were stimulated overnight with IL-15 (1ng/ml) and then co-incubated with target cells at 5:1 effector:target ratio for 4 hours. Golgistop (BD Biosciences) was added 1 hour after co-incubation and cells were then stained for surface markers REA147-FITC (Miltenyi Biotech), CH-L-PE (BD Biosciences), CD56-PE/Cy7 (Biolegend) and CD3-PerCP (Biolegend). Cells were then fixed and permeabilized, stained for IFNγ-BV421 (Biolegend) and TNFα-AF700 (Biolegend) and analysed by flow cytometry on a BD FACS Aria. Data was analysed by FlowJo_V10 software.

Blunt M.D., Pulido A.V., Fisher J.G., Graham L.V., Doyle A.D., Fulton R., Carter M.J., Polak M., Johnson P.W., Cragg M.S., Forconi F, & Khakoo S.I. (2022). KIR2DS2 expression identifies NK cells with enhanced anti-cancer activity. Journal of immunology (Baltimore, Md. : 1950), 209(2), 379-390.

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Multiparameter flow cytometry of NK cell subsets

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We used the following antibodies in this study: NKG2C-APC, NKG2C-PerCp, NKG2A-PE (R&D); CD3-FITC, CD4-APC-Cy7, CD16-PerCP-Cy5.5, CD56-PE-Cy7, CD107a-APC-H7 (BD Biosciences, San Jose, CA, USA); IFN-γ-BV421 (Biolegend, San Diego, CA, USA); CD4-FITC/CD8-PE/CD3-PerCP (BD Biosciences, San Jose, CA, USA).

Ma M., Wang Z., Chen X., Tao A., He L., Fu S., Zhang Z., Fu Y., Guo C., Liu J., Han X., Xu J., Chu Z., Ding H., Shang H, & Jiang Y. (2017). NKG2C+NKG2A− Natural Killer Cells are Associated with a Lower Viral Set Point and may Predict Disease Progression in Individuals with Primary HIV Infection. Frontiers in Immunology, 8, 1176.

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Murine Liver TIL Phenotyping by Flow Cytometry

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After isolation of the TILs from the liver, 100 μL volumes of cell suspension at a concentration of 1×10⁷ cells/mL were plated into designated wells of a 96-well plate. Cells were first stained with Live-Dead Aqua (Invitrogen) for 30 minutes on ice, washed twice with PBS, and then blocked with rat anti-mouse Fc antibody (CD16/CD32, BD Biosciences). After blocking, cells were incubated for 1 hour with the following anti-mouse fluorophores: CD3-PerCP-Cy5.5 (Biolegend), CD4-APC-Fire (BD Biosciences), CD8a-PE/Cy7 (Biolegend), PD-1-FITC (eBioscience), OX40-APC (Biolegend), CD44-PE (Biolegend), CD62L-APC (Biolegend), and CCR7-BV421 (Biolegend). Cells were washed following staining, and suspended in Fix/Perm buffer (eBioscience) for 30 minutes. After fixation, intracellular staining with Eomes PE (Invitrogen) and IFNγ BV421 (Biolegend) was done on ice for 30 minutes. Flow cytometry was performed using the CytoFLEX flow cytometer (B eckman Coulter).

Muth S.T., Saung M.T., Blair A.B., Henderson M.G., Thomas DL I.I, & Zheng L. (2020). CD137 Agonist-based Combination Immunotherapy Enhances Activated, Effector Memory T cells and Prolongs Survival in Pancreatic Adenocarcinoma. Cancer letters, 499, 99-108.

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