Draining lymph nodes (dLNs) and spleen were triturated and dispersed through a 40 μm strainer and then washed with PBS. After washing, single cell suspension was prepared for flow cytometry analysis [6 (link),33 (link)]. For surface staining, cells were stained by using anti-mouse CD3 (145-2C11, BD Bioscience, San Jose, CA, USA), CD4 (GK1.5, BD Bioscience), CD8 (53-6.7, BD Bioscience), CD11b (M1/70, BD Bioscience), CD11c (N418, BD Bioscience), and Gr-1 (RB6-BC5, BD Bioscience). For intracellular cytokine staining, cells were incubated for 4 hours with PMA (200 ng/mL, Merck, NJ, USA) and ionomycin (500 mg/mL, Merck) in the presence of Golgistop (BD Bioscience). After stimulation, cells were surface stained as described above. Then, cells were re-suspended with 250 μL fixation solution (BD Bioscience) and washed by permeabilization buffer (BD Bioscience). Twenty minutes later, cells were stained with intracellular antibodies, i.e., anti-IFN-γ (XMG1.2, eBioscience, San Diego, CA, USA), anti-IL-17A (TC11-18H10, BD Bioscience), anti-GM-CSF (MPI-22E9, Biolegend, San Diego, CA, USA), and anti-IL-10 (JES5-16E3, Biolegend) antibodies. For Ki-67 (SoIA15, eBioscience) staining, cells were fixed and permeabilized by transcription factor staining buffer set (eBioscience) for nuclear protein staining. Results were analyzed by FlowJo (Tree star Inc., Oakland, CA, USA).
Anti ifnγ xmg1.2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Anti-IFNγ (XMG1.2) is a monoclonal antibody that binds to and neutralizes interferon-gamma (IFN-γ). It is a laboratory research tool used for the detection and quantification of IFN-γ in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α mp6 xt22, by Thermo Fisher Scientific (7 mentions)
Anti il 17a ebio17b7, by Thermo Fisher Scientific (5 mentions)
Ionomycin, by Merck Group (4 mentions)
Golgiplug, by BD (4 mentions)
Golgistop, by BD (3 mentions)
Anti il 17a tc11 18h10, by BD (3 mentions)
Anti il 13 ebio13a, by Thermo Fisher Scientific (3 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (3 mentions)
Lsrfortessa, by BD (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Cd8 53 6, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti il 4 11b11, by BioLegend (2 mentions)
Fortessa, by BD (2 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (2 mentions)
Anti il 2 jes6 5h4, by Thermo Fisher Scientific (2 mentions)
Anti il 4 11b11, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
34 protocols using anti ifnγ xmg1.2
1
Multiparametric flow cytometry analysis of immune cells
2
Multiparametric Flow Cytometry Analysis
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The following combination of fluorescently labeled primary antibodies against cell surface markers and intracellular cytokines were used: anti-CD3 (17A2), anti-CD4 (clones GK1.5 and RM4-5), anti-CD8a (53–6.7), anti-CD44 (IM7), anti-CD45R/B220 (RA3-6B2), anti-CD11c (N418), anti-CD103 (M290), anti-Ly6C (HK1.4), anti-I-A/I-E (M5/114.15.2), anti-IL17 (TC11-18H10.1), and anti-IL4 (11B11) from Biolegend, and anti-IFN-γ (XMG1.2) and anti-IL-13 (eBio13A) from eBioscience. Cells were analyzed using a LSRII flow cytometer running FACSDiva Software (BD Bioscience) and analyzed using FlowJo Software (Tree Star). Fixable Viability Dye eFluor780 (eBioscience), DAPI or 7-AAD Viability Staining Solution (Biolegend) were used according to the manufacturer’s instructions to exclude dead cells from analysis.
3
Multiparametric Flow Cytometry Analysis
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Cells from lymph nodes and spinal cord were isolated as described before [26 ]. For detection of cell surface markers, cells were stained in FACS buffer (PBS containing 1% BSA and 0.1% NaN3) with the following fluorochrome labeled monoclonal antibodies: anti-CD45 (30-F11), anti-CD4 (RM4-5), anti-CD25 (PC61) and anti-CD44 (IM7). For intracellular cytokine staining, cells were incubated for 16 hours with anti-CD3 (0.5 μg/ml). Next, cells were fixed and permeabilized by incubation with Foxp3 Fixation/Permeabilization Buffer (eBioscience) and stained in Permeabilization Buffer (eBioscience) with the following fluorochrome labeled monoclonal antibodies: anti-Foxp3 (FJK-16s), anti-IL-17 (eBio17B7) and anti-IFNγ (XMG1.2). All antibodies were purchased from BD Pharmigen or eBioscience. For cell number quantification, 104 FACSuite FC Beads (BD) were added per sample prior to acquisition. Samples were acquired on FACS Verse (BD). FACS data were analyzed using FlowJo 7.6.5 software (TreeStar).
4
Multicolor Flow Cytometry Analysis
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Single cell suspension prepared from different organs were stained and acquired on FACSCanto (BD) or BD Fortessa and analyzed with FlowJo software (Tree Star). Surface phenotype staining was done with the following fluorochrome-conjugated or biotinylated mAbs: anti-CD3 (145-2c11), anti-CD4 (RM5), anti-ICOS (7E.17G9), anti-Thy1.1 (HIS51), anti-CD11c (HL3), anti-F4/80 (BM8), anti-CXCR3 (cxcr3-173), and anti-IFN-γR (2E2). The expression of Foxp3 (FJK-16s), T-bet (ebio4B10), CXCL9 (MIG-2F5.5) (eBioscience, San Diego, CA), CXCL10 (CL9177B) (Cedarlane, Burlington, ON), CXCL11 (rmCXCL11) (R&D systems, Burlington, ON), Ki-67 (B56) (BD Bioscience, Mississauga, Ontario) was determined by intracellular staining performed according to manufacturers’ protocols. The STAT1 phosphorylation assay was done following BD recommendations. To evaluate cytokine production, T cells were re-stimulated for 4hrs at 37°C with PMA (20ng/ml), ionomycin (1nM) (Sigma-Aldrich, Oakville, Canada) and BD GolgiStopTM (1:1000 dilution), and then stained intracellularly with anti-IFN-γ (XMG1.2) (eBioscience, San Diego, CA). T-bet fluorescence minus one (FMO) control and antibody titration is displayed in S1E Fig .
5
Multicolor Flow Cytometry Panel
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Fluorophore-conjugated antibodies against murine CD8a (53–6.7), CD62L (MEL-14), CD44 (IM7), PD-1 (29F.1A12), CTLA-4 (UC10-4B9), Tim-3 (RMT3-23), Lag-3 (C9B7W), CD25 (PC61), CD11c (N418), CD11b (M1/70), XCR1(ZET), CD80 (16-10A1), IL12 (C15.6), CD137L (TKS-1), CD215 (6B4C88), CCR7 (4B12), CXCR3 (CXCR3-173), as well as fluorophore-conjugated Streptavidin, TruStain FcX mouse anti-CD16/32 antibody (93) and reagents for life/dead discrimination 7-AAD and ZombieRed were all purchased from BioLegend. Anti-TNFα (TN3-19.12) and anti-IFNγ (XMG1.2) were purchased from eBiosciences. Anti-IL2 (JES6-5H4) and anti-CD212 (114) were purchased from BD Biosciences. Anti-iNOS2 (sc-7271) was purchased from Santa Cruz Biotechnology. PE-conjugated, peptide-loaded MHC class I tetramers were produced in-house as already described [21 (link)].
6
Multiparametric Flow Cytometry Analysis
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Cell surface markers were stained in 1×PBS containing 2% FBS with indicated antibodies. Anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD45.1 (A20), and anti-CD45.2 (104) were purchased from eBioscience. Intracellular staining was performed using the Foxp3/Transcription Factor Staining Kit (eBioscience) with indicated antibodies. For intracellular cytokine staining, cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich) and ionomycin (1 μg/mL; Sigma-Aldrich), and then treated for another 1 h with Golgi-Plug (BD Biosciences). Anti–IFN-γ (XMG1.2), anti-IL17A (eBio17B7), and anti–IL-4 (eBio 11B11) were purchased from eBioscience, and anti–cleaved caspase-3(Asp175) (D3E9), anti–phospho-H2A.x(Ser139) (20E3), and anti-LC3b (D11) were purchased from Cell Signaling Technology. All data collection was performed with a FACSAria II cell sorter (BD Biosciences), and data analysis was done with FlowJo software (Tree Star).
7
FACS Analysis of Immune Cell Subsets
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Anti-mouse antibodies (BioLegend unless otherwise noted) used for FACS analysis were as follows: FITC-conjugated anti-CD3 (17A2) and anti-CD4 (GK1.5; BD Pharmingen); PE-conjugated anti-CD4 (GK1.5), anti-CD19 (6D5), anti-CD69 (H1.2F3; BD Pharmingen), anti-IFN-γ (XMG1.2; eBioscience), anti-TNF-α (MP6-XT22; eBioscience), and anti-IL-2 (JES6-5H4; eBioscience); PE-Cy7-conjugated anti-NK-1.1 (PK136) and anti-CD44 (IM7; BD Pharmingen); APC-conjugated anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22) and anti-IL-7Rα (A7R34), and BV421-conjugated anti-CD62L (MEL-14).
8
Naive CD4+ T Cell Differentiation
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Naive CD4+ T (CD44lowCD62LhighCD25−) cells were purified using a CD4+ naive T cell negative isolation kit according to the manufacturer's protocol (STEMCELL Technologies). The in vitro differentiation experiments were performed as previously described. Naive CD4+ T cells were stimulated with immobilized anti-CD3 (5 μg/ml; 145-2C11; eBioscience) and anti-CD28 (5 μg/ml; 37.51; eBioscience) for 2 days. Then, the cells were washed and transferred to a new plate and further expanded in medium with hIL-2 (50 U/ml, R&D Systems) for 2 days. For Tfh-like cell differentiation, naive CD4+ T cells were activated with anti-CD3 and anti-CD28 as above and treated with 20 ng/ml IL-6 (R&D Systems), 20 ng/ml IL-21 (R&D Systems), 10 μg/ml anti-IL-4 (11B11, eBioscience), 10 μg/ml anti-IFN-γ (XMG1.2, eBioscience), and 20 μg/ml anti-TGF-β (1D11, R&D Systems) for 4 days.
9
Evaluating T helper cell responses in CIA mice
10
Multiparametric T Cell Immunophenotyping
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