ifnγ v450, by BD - Product details

1

Characterization of NK Cell Activation

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To assess proliferation and further characterisation of the differentiation of NK cells, PBMC were permeabilised and stained with anti-Ki67-PE (eBioscience, Hatfield, UK), Granzyme-B-FITC and Perforin-Cy5.5/PerCP (Biolegend, London, UK) directly ex-vivo. For intracellular staining for IFNγ production; PBMC were incubated with rhIL12 and rhIL15 (10ng/ml) (R&D systems, Abingdon, UK), for 19 hours at 37°C. 1mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 hours. Cells were then stained with anti-CD3-Cy5.5/PerCP or CD3/Pe-Cy7, CD16-APCy7, CD56-FITC, and subsequently fixed and permeabilised, followed by intracellular staining for IFNγ-v450 (BD Biosciences, Oxford, UK). Dead cells were excluded by fixable live dead stain.
For degranulation, PBMCs were incubated with K562 cells (5:1 E:T ratio) for 3 hours following overnight stimulation with a combination of 50ng/ml rhIL12 and rhIL18 (Miltenyi Biotech). Anti-CD107a-PE mAb (BD Biociences, Oxford, UK) was added at the time of stimulation with target-cells and monensin (1mM) added during the last 2 hours of incubation prior to staining and acquisition.

Gill U.S., Peppa D., Micco L., Singh H.D., Carey I., Foster G.R., Maini M.K, & Kennedy P.T. (2016). Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo. PLoS Pathogens, 12(8), e1005788.

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2

Multiparameter Flow Cytometry Analysis

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For surface staining, cells were stained with surface markers for 30
min on ice and washed twice with FACS buffer (PBS with 2% FBS and 2
mM EDTA buffer). After, cells were fixed and permeabilized with Fix/Perm
buffer (BD Biosciences) for 20 min on ice, washed twice with BD Perm/Wash,
and stained with the intracellular antibodies for 60 min on ice. For PoxP3
staining, cells were fixed and permeabilized with Fix/Perm Buffer
(eBiosciences, San Diego, CA, USA) for 60 min on ice, washed twice with
Perm/Wash buffer (eBiosciences) and stained with intracellular antibodies
for 60 min on ice. Subsequently, the cells were washed twice with the
respective Perm/Wash buffer and kept in 2% paraformaldehyde.
Antibodies used: PBS57-CD1d tet APC (kindly donated by NIH tetramer resource
facility), Vα24 FITC, and CD3 ECD were from Beckman Coulter
(Fullerton, CA), CD4 Qdot655, CD8 Qdot 605, and the viability marker AmCyan
were from Life Technologies (Carlsbad CA, USA), CD25 APC, CD38 PE, HLA-DR
PerCP, IFNγ V450, TNF Alexa700, IL-10 PE, and IL-4 PE-Cy7 were all
from BD bioscience. Data were acquired on a BD LSRFortessa instrument (BD
Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar,
Ashland, OR, USA).

Paquin-Proulx D., Ching C., Vujkovic-Cvijin I., Fadrosh D., Loh L., Huang Y., Somsouk M., Lynch S.V., Hunt P.W., Nixon D.F, & SenGupta D. (2016). Bacteroides are associated with GALT iNKT cell function and reduction of microbial translocation in HIV-1 infection. Mucosal immunology, 10(1), 69-78.

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3

Cytokine Production in PBMCs

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For intracellular staining for IFN-γ production, PBMCs were incubated with 50 ng/ml of rhIL-12 (Miltenyi Biotec) and 50 ng/ml of rh-IL18 (R&D Systems) for 21 h at 37°C. One mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 h. Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) and subsequently fixed and permeabilized, followed by intracellular staining for IFN-γ-V450 (BD Biosciences). Dead cells were excluded by live/dead stain (Invitrogen).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).

Heiberg I.L., Pallett L.J., Winther T.N., Høgh B., Maini M.K, & Peppa D. (2015). Defective natural killer cell anti-viral capacity in paediatric HBV infection. Clinical and Experimental Immunology, 179(3), 466-476.

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4

Modulating T Cell Activation in CLL

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T cells were magnetically purified from PBMC using a PAN T-cell isolation kit (Miltenyi Biotec) from healthy donors. Healthy donor T cells (1 × 10⁵) were then cultured with bead-purified (Miltenyi Biotec) CD19+ B cells from CLL patients (1 × 10⁶) at an optimal 1:10 ratio per well in the presence or absence of CXCL12 (250 ng/ml), lenalidomide (10 µM), CXCR4, or mouse anti-human IL-10 blocking antibodies (2 µg/ml, R&D). Each experiment was performed in duplicate. After 48 h of culture, T cells were magnetically purified again as described above (purity >95%) and activated with anti-CD3/CD28 magnetic Dynabeads (Invitrogen) for 6 h. A negative control (no stimulation) was included in every experiment. Brefeldin A (10 µg/mL) and CD107a PE-CF594 (BD Biosciences) were added to the culture. Cells were stained with a Live/Dead-Aqua (Invitrogen), CD3-BV650, CD8-FITC, CD4-APC-Cy7, fixed/permeabilized (BD Biosciences) followed by intracellular staining with IFN-γ-V450, TNF-α-Alexa 700, and IL-2-PE (BD Biosciences).

Shaim H., Estrov Z., Harris D., Hernandez Sanabria M., Liu Z., Ruvolo P., Thompson P.A., Ferrajoli A., Daher M., Burger J., Muftuoglu M., Imahashi N., Li L., Liu E., Alsuliman A.S., Basar R., Nassif Kerbauy L., Sobieski C., Gokdemir E., Kondo K., Wierda W., Keating M., Shpall E.J, & Rezvani K. (2018). The CXCR4–STAT3–IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide. Frontiers in Immunology, 8, 1773.

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5

Phenotyping Antigen-specific T Cells

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10⁸/ml of electroporated T cells were stained with 1 μM of CFSE (ThermoFisher #C34554) for 5mins in the dark before being washed in HBSS supplemented in 2% FBS. 2 μg/ml of Brefeldin A, CFSE engineered T cells, and HepG2.2.15 were spiked into whole blood. The spiked blood was next incubated at 37°C and at eight revolutions per minute for 16 hours using the Amersham biosciences hybridisation oven/shaker. Red blood cells were then lysed with Red Blood Cell Lysis solution (Miltenyi Biotec), and remaining cells were stained with LIVE/DEAD™ Fixable Yellow Dead Cell Stain (ThermoFisher #L34968) before being blocked with Human TruStain FcX™ (Biolegend #422301) and stained with CD3 PerCP-Cy5.5 (BD Biosciences #340949), CD8 Pe-Cy7 (BD Biosciences #557746) antibodies. Cells were then fixed and permeabilised using Cytofix/Cytoperm™ Fixation and Permeabilisation Solution (BD) before being stained with IFNγ V450 (BD Biosciences #560371), TNFα APC (BD Biosciences #554514) antibodies.

Lin M., Bhakdi S.C., Tan D., Lee J.J., Tai D.W., Pavesi A., Wai L.E., Wang T., Bertoletti A, & Tan A.T. (2023). Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood. Immunotherapy Advances, 3(1), ltad015.

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6

T Cell Cytokine Production Profiling

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For cytokine production analysis by T cell subsets, surface staining was first performed with CD45-PERCP, CD3-FITC, and CD8-APC-H7 antibodies (BD Biosciences). Samples were then lysed, fixed, permeabilized, and then stained with IL-17A-eFluor660 (eBiosciences), IFNγ-V450, or IL-5-APC or IL-21-PE (BD Biosciences) antibodies. Samples were analyzed by flow cytometry.

Braudeau C., Néel A., Amouriaux K., Martin J.C., Rimbert M., Besançon A., Giraudet S., Terrien C., Aliaga M., Salabert-Le Guen N., Hémont C., Hamidou M, & Josien R. (2017). Dysregulated Responsiveness of Circulating Dendritic Cells to Toll-Like Receptors in ANCA-Associated Vasculitis. Frontiers in Immunology, 8, 102.

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7

Multiparametric Flow Cytometry Analysis

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Following incubation, EDTA (Hoefer) was added at 20 mM to arrest stimulation. Cells were surface stained with LIVE/DEAD Fixable Yellow Stain (Life Technologies), fixed and permeabilized with FACS Lysing Solution and FACS Permeabilizing Solution II (BD Biosciences), and stained with the following intracellular antibodies: CD3-PE (UCHT1, Biolegend), TNF-FITC (MAb11, BD Biosciences), IFNγ-V450 (B27, BD Biosciences), CD16-PerCP Cy5.5 (3G8, Biolegend). For degranulation, cytokine production, and ADCC experiments, cells were also surface stained with CD56-PE-Cy7 (B159, BD Biosciences). For cytotoxicity experiments, no additional antibodies were added. Stained cells were analyzed on the MACSQuant Analyzer (Miltenyi Biotec). Analysis and compensation were performed using FlowJo (FlowJo, LLC).

Strauss-Albee D.M., Liang E.C., Ranganath T., Aziz N, & Blish C.A. (2016). The Newborn Human NK Cell Repertoire is Phenotypically Formed but Functionally Reduced. Cytometry. Part B, Clinical cytometry, 92(1), 33-41.

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8

Multiparameter Flow Cytometry Analysis

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For surface staining, cells were stained with surface markers for 30
min on ice and washed twice with FACS buffer (PBS with 2% FBS and 2
mM EDTA buffer). After, cells were fixed and permeabilized with Fix/Perm
buffer (BD Biosciences) for 20 min on ice, washed twice with BD Perm/Wash,
and stained with the intracellular antibodies for 60 min on ice. For PoxP3
staining, cells were fixed and permeabilized with Fix/Perm Buffer
(eBiosciences, San Diego, CA, USA) for 60 min on ice, washed twice with
Perm/Wash buffer (eBiosciences) and stained with intracellular antibodies
for 60 min on ice. Subsequently, the cells were washed twice with the
respective Perm/Wash buffer and kept in 2% paraformaldehyde.
Antibodies used: PBS57-CD1d tet APC (kindly donated by NIH tetramer resource
facility), Vα24 FITC, and CD3 ECD were from Beckman Coulter
(Fullerton, CA), CD4 Qdot655, CD8 Qdot 605, and the viability marker AmCyan
were from Life Technologies (Carlsbad CA, USA), CD25 APC, CD38 PE, HLA-DR
PerCP, IFNγ V450, TNF Alexa700, IL-10 PE, and IL-4 PE-Cy7 were all
from BD bioscience. Data were acquired on a BD LSRFortessa instrument (BD
Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar,
Ashland, OR, USA).

Paquin-Proulx D., Ching C., Vujkovic-Cvijin I., Fadrosh D., Loh L., Huang Y., Somsouk M., Lynch S.V., Hunt P.W., Nixon D.F, & SenGupta D. (2016). Bacteroides are associated with GALT iNKT cell function and reduction of microbial translocation in HIV-1 infection. Mucosal immunology, 10(1), 69-78.

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Ifnγ v450

Lab products found in correlation

8 protocols using ifnγ v450

Characterization of NK Cell Activation

Multiparameter Flow Cytometry Analysis

Cytokine Production in PBMCs

Modulating T Cell Activation in CLL

Phenotyping Antigen-specific T Cells

T Cell Cytokine Production Profiling

Multiparametric Flow Cytometry Analysis

Multiparameter Flow Cytometry Analysis

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