For tissues of ID8 xenografts, tumor tissues were cut into small pieces and digested using a tumor dissociation kit (Miltenyi Biotec; cat #4035581) according to the instructions, on a shaking table for 1 h. A 200-mesh stainless steel filter was used to remove the remaining mass of tissue. After centrifugation (300 g, 10 min), single immune cells were collected and resuspended in stain buffer (BD Biosciences; cat #4032357). Samples were either directly incubated with the following antibodies from BD Biosciences: CD45/cat #557659, CD3/cat #555275, CD4/cat #563151, CD8a/cat #553030, CD11c/cat #558079, CD279/cat #744546, CD274/cat #741014, MHCII/cat #746197; or incubated with Foxp3/cat #560401, ki-67/cat #561283, granzyme B/Ebioscience/cat #46-8898-82, or IFN-γ/BD Biosciences/cat #563376 after permeabilization in the dark at room temperature for 40 min. As for IFN-γ detection, the single cells were stimulated with Leukocyte Activation Cocktail (BD Biosciences; cat #550583) for 6 h before being incubated with IFN-γ antibodies. Beckman Coulter flow cytometry (Cytoflex LX) was used to analyze the components of immune cells in the tumor, and FlowJo V10 software was used to quantify populations.
Ifn γ
Manufactured by BD
Sourced in United States, United Kingdom, Germany, China
IFN-γ is a cytokine that plays a crucial role in the immune system. It is produced by various cell types, including T cells and natural killer cells, in response to antigenic or mitogenic stimulation. IFN-γ is involved in the regulation of immune responses and the control of viral infections.
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Lab products found in correlation
Tnf α, by BD (87 mentions)
Interleukin 4 (il 4), by BD (65 mentions)
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Interleukin 6 (il 6), by BD (37 mentions)
Interleukin 2 (il 2), by BD (35 mentions)
Il 17, by BD (29 mentions)
Il 1β, by BD (24 mentions)
Il 17a, by BD (21 mentions)
Foxp3, by BD (18 mentions)
Golgiplug, by BD (18 mentions)
Ionomycin, by Merck Group (18 mentions)
Brefeldin a, by Merck Group (16 mentions)
Il 17a, by Thermo Fisher Scientific (15 mentions)
Cd11b, by BD (14 mentions)
Foxp3, by Thermo Fisher Scientific (14 mentions)
Phorbol myristate acetate (pma), by Merck Group (14 mentions)
Il 13, by BD (13 mentions)
Golgistop, by BD (13 mentions)
Facscalibur, by BD (13 mentions)
Tnf α, by R&D Systems (11 mentions)
389 protocols using ifn γ
1
Immune Cell Profiling in Murine Tumor
2
Isolation and Stimulation of Colonic Lamina Propria Mononuclear Cells
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Colonic lamina propria mononuclear cells (cLPMNCs) were isolated in accordance with an established protocol.(7 (link),9 (link),11 (link)) cLPMNCs (2 × 106/ml) were stimulated with Staphylococcus aureus (SAC, 1:10,000; Calbiochem, La Jolla, CA) and IFN-γ (50 ng/ml; BD Biosciences, San Jose, CA) to induce IL-12/23 p40 production.(7 (link)) cLPMNCs were also cultured with anti-CD3 monoclonal Ab (5 μg/ml; BD Biosciences) for IFN-γ production.(9 (link),18 (link)) Cells were cultured in complete RPMI medium for 60 h. Cytokine levels in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA).
3
Characterization of Adoptive T Cell Responses
4
Comprehensive Immune Cell Profiling
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Ab’s for cell purification and flow cytometry were as follows: CD16/CD32 (Fc Block; BD), TCR Vα2 (BioLegend), CD4 (BioLegend), CD25 (BioLegend), CD69 (BioLegend), CD19 (BioLegend), Foxp3 (BD), IL-17A (BD), IFN-γ (BD), Ki67 (BD), TCR γδ (BioLegend), NK-1.1 (BioLegend), CD49b (BioLegend), CD8a (BioLegend), CD11c (BioLegend), Siglec H (BioLegend), PDCA-1 (BioLegend), B220 (BioLegend), IL-5 (BD), IFN-γ (BD), CD28 (Bioscience), CD3 (eBioscience), and anti–human IgG-Fc (SouthernBiotech). Intracellular cytokine staining was done with the Cytofix/Cytoperm kit (BD). T cell proliferation was assessed by flow cytometry using an anti–human Ki67 staining kit (BD). Foxp3 staining was performed with an eBioscience kit. Flow cytometry was performed with FACS CyAn (Beckman Coulter) and analyzed with FlowJo software.
5
SARS-CoV-2 Specific T-cell ELISpot Assay
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ELISpot plates (MilliporeSigma) were coated with either IFNγ (BD Biosciences, #51-2525KZ), IL-17A (Invitrogen, #88-7371-88), or IL-5 (BD Biosciences, #51-1805KZ) capture antibodies at a 1:200 dilution in ELISpot coating buffer (eBioscience, Waltham, MA). After an overnight incubation at 4 o C, plates were washed and then blocked with complete RPMI (cRPMI) medium for at least 2 hours. Splenocytes harvested as described above were plated at 2 x 10 5 cells per well. A subset of each sample was stimulated with PepMix SARS-CoV-2 (JPT Peptide Technologies, #PM-WCPV-S-1) at a final concentration of 1 µg/mL. Plates were then incubated at 37°C and 5% CO2 for 48 hours. After a wash with PBS with 0.1% Tween 20, 100 µL of detection antibody (IFNγ, BD Biosciences, #51-1818KA; IL-17A, Invitrogen, #88-7371-88; and IL-5, BD Biosciences, #51-1806KZ) was added at a 1:250 dilution in ELISpot diluent (eBioscience) overnight at 4 o C. Plates were washed and developed with Vector NovaRED Substrate Peroxidase (Vector Laboratories, Burlingame, CA; #SK-4800) for 15 minutes. The reaction was stopped by washing the plates with deionized water, and plates were left to dry in the dark. Spots were counted and data were analyzed using ImmunoSpot software (Cellular Technology Limited, Cleveland, OH).
6
Murine Dendritic Cell Isolation and Stimulation
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Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), propidium iodide (PI), and ovalbumin (OVA) were purchased from Sigma-Aldrich (Steinheim, Germany), and lipopolysaccharide (LPS) and OVA-Alexa 488 were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE-conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA): FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), IA[b] (AF6–120.1), IFN-γ, CD4, PE-CD8, CD4. Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-2, IL-10, and TNF-α were purchased from BD Biosciences (San Jose, CA, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III and collagenase D were purchased from Roche (Salt Lake City, UT, USA).
7
Immunophenotyping of Human T Cells
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Human CD3 (BD Bioscience), CD4 (BD Bioscience), TCRgd (BD Bioscience), TCR Vd1 (Invitrogen), TCR Vδ2 (Invitrogen), IL-17A (BD Bioscience), and IFN-γ (BD Bioscience) mAbs were purchased from BD bioscience or Thermo Fisher Scientific. In brief, for cell surface staining, 0.5 × 105 cells/100 μl staining buffers were incubated with a cocktail for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized (BD Bioscience) and then stained intracellularly for IL-17A and IFN-γ. Flow cytometry analysis was performed on BD FACS Calibur (BD Bioscience) and analyzed with FlowJo software (Flowjo LLC, Ashland, OR).
8
Splenocyte-BMDC Co-culture Cytokine Assay
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Spleens were aseptically removed and single cell suspensions were obtained as described in Section 2.2.4 . Splenocytes (5 × 106) were co-cultured with BMDC (5 × 105) either or not loaded with Influvac in U-bottom 96-well plates at 37 °C, 5% CO2. After 5 days of incubation, supernatants were collected and stored at −20 °C for cytokine analysis. IFNγ, IL-13 (both from R&D) were measured by means of ELISA according to the protocol described in Section 2.1.4 . for galectin-4 and -9. Purified rat anti-mouse antibodies (1 µg/mL for IFNγ and 2 µg/mL for IL-13), recombinant mouse cytokines and biotinylated rat anti-mouse antibodies (1 µg/mL for IFNγ and 400 ng/mL for IL-13) were purchased from BD Biosciences. TNFα (Biolegend), IL-10 and IL-17A concentrations (both from Thermo fisher) were measured by ELISA according to manufacturer’s protocol.
9
Multiparametric Flow Cytometry Analysis
10
Cytokine Detection in Lymphocytes
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Spleen or lymph node cells were stimulated with 2.5 µg/ml of Cry j 2 and supernatants were collected at 72 h and stored at −20 °C for cytokine detection assays. The concentrations of IL-5, IL-13, and IFN-γ were determined using standard sandwich ELISA protocols (IL-5: BD Biosciences, NJ, USA; IL-13: Thermo Fisher Scientific Inc.; IFN-γ: BD Biosciences).
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