LPLs and pLN cells stimulated for 4 hours with PMA (50 ng/ml; Sigma), ionomycine (1 μg/ml; Sigma), and the Golgi-traffic inhibitor Brefeldin (1 μl/ml; BD Biosciences). Cells were stained with anti-CD3-PE (BD Pharmingen) or anti-CD3-APC (eBioscience) and anti-CD4-APC (Biolegend) or anti-CD4-FITC (BD Pharmingen). Next, the cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience). For intracellular staining the cells were incubated in permeabilization buffer (eBioscience) containing anti-IL-17-FITC (Biolegend), anti-IFNγ-FITC (BD Pharmingen), anti-IL-4-PE (BD Pharmingen) or Foxp3-FITC (eBioscience). An appropriate isotype matched control antibody was used in all FACS analyses. Cells were analyzed on a FACS Calibur using the CellQuest software (BD Biosciences). Results were analyzed with FlowJo version 7.6.5.
Anti ifn γ fitc
Manufactured by BD
Sourced in United States, Australia, Germany
Anti-IFN-γ-FITC is a fluorescent-labeled antibody that binds to interferon-gamma (IFN-γ). It is used in flow cytometry applications for the detection and quantification of IFN-γ-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (12 mentions)
Brefeldin a, by Merck Group (10 mentions)
Golgistop, by BD (8 mentions)
Anti il 4 pe, by BD (7 mentions)
Phorbol myristate acetate (pma), by Merck Group (7 mentions)
Golgiplug, by BD (7 mentions)
Anti mip1β pe, by BD (6 mentions)
Brefeldin a, by BD (5 mentions)
Anti cd16 apc cy7, by BD (4 mentions)
Anti cd56 pe cy7, by BD (4 mentions)
Anti cd3 pacific blue, by BD (4 mentions)
Anti il 17 fitc, by BioLegend (3 mentions)
Anti cd4 apc, by BioLegend (3 mentions)
Cellquest software, by BD (3 mentions)
Phorbol myristate, by Merck Group (3 mentions)
Rosettesep nk cell enrichment kit, by STEMCELL (3 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (3 mentions)
Anti cd3 apc, by BD (3 mentions)
Anti cd4 percp, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
52 protocols using anti ifn γ fitc
1
Characterization of Th17 and Treg Cells
2
Multiparametric Flow Cytometry Analysis
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Production of IFNγ and TNFα by T, NKT-like and NK cells was determined on blood and BAL samples from all subjects as previously reported [11 (link)–13 (link)].
Briefly, one mL blood diluted 1:2 in RPMI and prepared BAL cells were stimulated with phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma). Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma) and the tubes re-incubated in a humidified 5% CO2/95% air atmosphere at 37°C for 16 h. Aliquots of blood and BAL were added to FACS tubes (BD) and treated with FACSLyse and FACSPerm as above and five μL of appropriately diluted anti- IFNγ FITC (BD), CD3 perCP.CY5.5 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), TNFα V450 (BD) and CD45 V500 (BD) monoclonal antibodies were added for 15 min in the dark at room temperature. Two mL of 0.5% bovine serum albumin (Sigma) / Isoflow (Beckman Coulter) was then added and the tubes centrifuged at 300 ×g for 5 min. After decanting, cells were analyzed as above.
Briefly, one mL blood diluted 1:2 in RPMI and prepared BAL cells were stimulated with phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma). Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma) and the tubes re-incubated in a humidified 5% CO2/95% air atmosphere at 37°C for 16 h. Aliquots of blood and BAL were added to FACS tubes (BD) and treated with FACSLyse and FACSPerm as above and five μL of appropriately diluted anti- IFNγ FITC (BD), CD3 perCP.CY5.5 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), TNFα V450 (BD) and CD45 V500 (BD) monoclonal antibodies were added for 15 min in the dark at room temperature. Two mL of 0.5% bovine serum albumin (Sigma) / Isoflow (Beckman Coulter) was then added and the tubes centrifuged at 300 ×g for 5 min. After decanting, cells were analyzed as above.
3
Quantifying IFN-γ-secreting CD8+ T Cells
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Antibodies used in the current study were Anti-CD11b PE (eBioscience M1/70)/APC (BD,M1/70), anti-GR-1 FITC (BD RB6-8C5), anti-Ly6G FITC (RB5-8C5) APC (RB5-8C5), anti-Ly6C PreCP-Cr™5.5 (BD AL-21), anti-IL10 FITC (BD), anti-IL12 (P40/P70) (BD), anti-p-Stat3 PE (BD PY705), anti-NOS2 PE (eBioscience CXNFT), arginase FITC (R&D), anti-CD8 PE (BD), anti-IFNγ FITC (BD).
The numbers of IFN-γ-secreting CD8+ T cells were analyzed by flow cytometry after adding Golgi plug (1 μg/mL, BD Pharmingen) for 8 hours. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton-Dickinson Immunocytometry System, Mountain View, CA) and Flowjo 10 software.
The numbers of IFN-γ-secreting CD8+ T cells were analyzed by flow cytometry after adding Golgi plug (1 μg/mL, BD Pharmingen) for 8 hours. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton-Dickinson Immunocytometry System, Mountain View, CA) and Flowjo 10 software.
4
Intracellular Cytokine Staining of CD4+ T Cells
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The in vitro differentiated CD4+ T cells were stimulated using an immobilized anti-TCR-β mAb (3 µg/ml, H57–597, BioLegend) for 6 h in the presence of monensin (2 µM). The cells were fixed with 4% paraformaldehyde (Cat#163–20145, Wako) and permeabilized with permeabilization buffer (50 mM NaCl, 5 mM EDTA, 0.02% NaN3, and 0.5% Triton X-100). Then, the cells were stained using the following antibodies, anti-IL-4-PE (Cat#504103, BioLegend, 1:50), anti-IL-5-APC (Cat#504305, BioLegend, 1:50), anti-IL-13-PE (Cat#12–7133–41, eBiosience, 1:50), anti-IFN-γ-FITC (Cat#562019, BD Biosciences, 1:500), and anti-IL-2-APC (Cat#503809, BioLegend, 1:50). For the intracellular staining of Gata3, the Foxp3/Transcription Factor Staining Buffer Kit (cat#TNB-0607, TONBO) was used according to the manufacturer’s protocol. Flow cytometry was performed using a Gallios Flow Cytometer instrument (Beckman Coulter) and a FACS Caliber instrument (BD Biosciences) and the results were analysed using the FlowJo software program (Tree Star, Ashland, OR, USA).
5
Progesterone Modulates PBMC and NK Cell Function
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Peripheral blood mononuclear cells (PBMCs) from healthy individuals and patients (obtained from fresh blood and later frozen) were cultured in a 96‐well plate. Cells were stimulated with PMA (50 ng/mL) and ionomycin (400 ng/mL, both from Sigma) for 1 hour, with or without progesterone 1 or 10 μg, or progesterone 10 μg alone (Sigma Aldrich), at 37°C. Brefeldin A was added and the cells were incubated for another 4 hours. Samples were fixed, permeabilized and stained with IL‐2 APC, CD4 PE‐Cy7, CD8 eFluor450, CD3 FITC (all eBioscience). Cytokine‐producing cells were detected by flow cytometry (FACS Canto‐II, BD). NK cell stimulation: PBMCs from healthy individuals were cultured in a 24‐well plate. Cells were stimulated with IL‐12 and IL‐18 alone or with pre‐treatment with progesterone 1 or 10 μg, or progesterone 10 μg alone. After 18 hours, brefeldin A was added to the cultures and the cells were incubated for an additional 4 hours. Cellular activation and surface markers were measured using anti‐CD3 PacificBlue (OKT3, eBioscience), anti‐CD56 PE (MY31, BD) and anti‐CD69 APC (L78, BD), followed by fixation with 2% formaldehyde, and permeabilization with anti‐IFN‐γ FITC (BD). Activated and cytokine‐producing NK cells were assessed by flow cytometry and analysed using FlowJo version 10.1 (Tree Star Inc).
6
NK Cell Functional Assay with Immune Complexes
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Human NK cells were isolated from buffy coats using RosetteSep NK cell enrichment kit (StemCell Technologies) and Ficoll separation. The isolated NK cells were rested overnight at 1.5 × 106 cells/mL in IL-15 at 37°C. ELISA plates were coated with antigen at 300 ng/well and incubated for 2 hours at 37°C. Plates were blocked with 5% BSA in PBS overnight at 4°C. The next day, 100 μL of antibodies, at a concentration of 5 μg/mL, were added to the plates. Plates were incubated for 2 hours at 37°C to form immune complexes. After the incubation, NK cells were added to the plates at 5 × 104 cells/well in R10 supplemented with anti-CD107a PE-Cy5, Brefeldin A (MilliporeSigma, B7651-5MG), and GolgiStop (BD Biosciences, 555802). Plates were incubated for 5 hours at 37°C. Following the incubation, NK cells were stained for the surface markers with anti-CD56 PE-Cy7, anti-CD16 APC-Cy7, and anti-CD3 Pacific Blue (BD Biosciences, 557747, 557758, 558124). NK cells were fixed and permeabilized with Fix&Perm cell permeabilization kit (Invitrogen, Thermo Fisher Scientific). Cells were incubated with anti–MIP-1β PE and anti–IFN-γ FITC (BD Biosciences, 550078, 340449) to stain for intracellular markers. Cells were acquired on an Intellicyt iQue.
7
Flow Cytometry Staining Reagents
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Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).
8
Intracellular Cytokine Profiling of Activated PBMCs
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PBMCs were stimulated with anti-CD3 antibody (0.1 μg/ml) for 6 h. After 1 h of incubation, brefeldin A and monensin (BD Biosciences) were added to stimulate intracellular cytokine protein accumulation. Following surface staining with anti-CD3-PE-Cy7, anti-CD4-Horizon V500, anti-CD8-APC-Cy7, anti-CD31-FITC, anti-CXCR4-PerCP-Cy5.5, and anti-CD28-APC-H7, the cells were fixed and permeabilized using the Fixation/Permeabilization Buffer Kit and further stained for intracellular cytokines with anti-TNF-α-FITC, anti-IFN-γ-FITC, anti-IL-6-PE, and anti-IL-10-APC (all from BD Biosciences).
9
Multiparametric Flow Cytometry Analysis
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After blocking with anti-FcR (CD16/32, BD bioscience) for 20 min, cells were incubated with specific mAbs at 4°C for 30 min. The following mAbs were used: anti-mouse CD3e-APC-Cy7; anti-CD4-PE-Cy7; anti-NK1.1-APC; anti-CD11b-PE-Cy7; anti-CD11cFITC; 7-AAD; anti-PDCA-1-APC; anti-CCR9-PE; anti-IFN-γ-FITC; and anti-IL-17-APC (eBioscience, BD bioscience). Background fluorescence was assessed by staining with irrelevant anti-rat isotypes (BD bioscience). Stained cells were analyzed by flow cytometry (FACS Canto II, Becton Dickinson Co.) and data analyzed using FlowJo software (Tree Star Inc.) [12] (link).
10
Cytokine Profiling of Activated PBMCs
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PBMCs were stimulated with anti-CD3 antibody (100 ng/ml) for 6 h. Then, after 1 h of incubation, brefeldin A (GolgiPlug, BD Biosciences) and monensin (GolgiStop, BD Biosciences) were added to the culture to cause intracellular cytokine accumulation. Cells were surface-stained with anti-CD3-Horizon V500, anti-CD4-PE-Cy7, anti-CD8-APC-H7, anti-CD28-APC, and anti-CD57-Pacific blue. Then, cells were fixed and permeabilized with the Fixation/Permeabilization Buffer Kit (BD Biosciences). Cells were then stained to detect intracellular cytokines with anti-TNF-PE-Cy7 and anti-IFN-γ-FITC (all from BD Biosciences). FACS analysis was performed with an LSR II Flow Cytometer, and data were analysed with FlowJo software.
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