For the identification of CD4+ Th cell subsets and CD8+ Tc cells, we used cytoplasmic cytokine staining method. Briefly, whole blood were diluted to 1:1 with saline solution and incubated with phorbol-12-myristate 13-acetate (PMA) (25 ng/ml), ionomycin (1 μg/ml) and Golgi Stop brefeldin A (10 μg/ml) (all from Sigma Aldrich, St. Louis, Missouri, USA) for 5 h at 37°C in 5% CO2 milieu. The following monoclonal antibodies were used for cell surface staining: CD4-PE-Cy5 or CD8-PE-Cy5 (both from Beckman Coulter). The cells were then fixed and permeabilized with Intraprep™ permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions, and intracellular cytokines were stained with the combination of interferon (IFN)-γ-FITC, interleukin (IL)-4-PE, IL-10-PE (all from BD Biosciences) and IL-17-PE (R&D Systems, Minneapolis, MN, USA) monoclonal antibodies. Measurements were performed and data were analyzed on Coulter FC500 flow cytometer (Beckman Coulter) equipped with Kaluza 1.2a software. IgG1-FITC (BD Biosciences) and IgG1-PE (R&D Systems) isotype-matched antibodies were used during the identification. Cells were quantified as their percentage in the CD4+ or CD8+ lymphocyte population.
Ifn γ fitc
Manufactured by BD
Sourced in United States
IFN-γ-FITC is a fluorescently labeled antibody that binds to the cytokine interferon-gamma (IFN-γ). It is used in flow cytometry applications to detect and quantify IFN-γ-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (13 mentions)
Cytofix cytoperm, by BD (6 mentions)
Il 17 pe, by BD (5 mentions)
Brefeldin a, by Merck Group (5 mentions)
Cd4 percp, by BD (5 mentions)
Cd3 percp, by BD (5 mentions)
Il 10 pe, by BD (5 mentions)
Facscalibur, by BD (5 mentions)
Brefeldin a, by BD (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Cd3 apc, by BD (4 mentions)
Cd4 pe cy7, by BD (4 mentions)
Cd8 apc h7, by BD (4 mentions)
Cd4 pe, by BD (4 mentions)
Golgistop, by BD (4 mentions)
Facsverse, by BD (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Il 4 apc, by BD (3 mentions)
Cd28 pe cy7, by BD (3 mentions)
Il 2 percp cy5, by BD (3 mentions)
64 protocols using ifn γ fitc
1
Flow Cytometric Identification of Lymphocyte Subsets
2
Isolation and Activation of Naïve T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Naïve T cells from PBMCs were isolated using a Miltenyi Biotec (Alburn, CA) negative selection kit. Purified naïve CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml, BD Bioscience, San Jose CA), soluble anti-human CD28 (1 μg/ml, BD Bioscience), and IL-2 (20 ng/ml, R&D Systems) with or without FTY720 (100 ng/ml, Novartis). After 6 days, cell-free culture supernatants were collected for cytokine analysis by Luminex assay (Miltenyi Biotec), and cells were harvested for RNA extraction and intracellular staining. Naïve T cells were stimulated with PMA (Sigma), ionomycin (Sigma), and Golgistop for 4 h. Cells were stained for anti-human CD4 APC (BD Bioscience) and violet fluorescent reactive dye VVD (Life Technologies), and then cells were fixed and permeabilized with BD fixation and permeabilization buffer and stained for IFN-γ FITC and GZMB FITC (BD Bioscience). For surface staining, the following antibodies were used: anti-human CD4 pacific blue, anti-human CCR7 PE, and anti-human CD45RA APC, IgG2a PE isotype control, IgG2b, and k APC isotype control (all from BD Bioscience). All antibodies were titrated for flow cytometry, which was performed on a BD LSR II (BD Bioscience) and analyzed using Flowjo software.
3
Profiling Lung T Cell Activation and Cytokines
4
Tumor-Infiltrating Lymphocyte Characterization
5
Cytokine and Immune Cell Profiling
6
CFSE Proliferation Assay with Cytokine Stimulation
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CFSE staining was done using 5 µM CFSE (Invitrogen, Cat# C34554) as described by Parish et al. [42] (link) and stimulated with cytokines for 6 days prior to flow cytometric analysis. Antibodies used: CD8-APC (BD, Cat# 345775), CD3-APC (BD, Cat# 555335), CD4-APC (Biolegend, Cat# 300514), CD56-APC (eBioscience, Cat#17-0569-42), CD16-PE (Biolegend, Cat#302007), HLA-DR-PE (Biolegend, Cat# 307605), NKG2D-APC (RnD Systems, Cat# FAB139A). For intracellular staining, IL-6-PE (eBioscience, Cat#12-7069) and IFN-γ-FITC (BD, Cat# 554700) were used. Isotype controls were IgG1 APC (BD, cat # 555751), rat IgG1-PE (Invitrogen, Cat # R104) and IgG1-FITC (BD, Cat #555748). PBMCs were incubated for 6 h +/− LPS (Sigma-Aldrich, L2654, 1 µg/mL) and cells were stained using the BD Cytofix/Cytoperm Kit (BD, Cat# 554714), Golgistop (BD, Cat# 554724) or Golgiplug (BD, Cat# 555029) according to the manufacturer’s protocol. Samples were run on an Accuri C6 Flow cytometer and data were analyzed using FCS Express v. 3.
7
Multiparameter Flow Cytometry Analysis of T Cells
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The remaining blood was used to isolate PBMCs using previously described methods [25] (link). PBMCs were thawed, washed and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen), and then stained with the following fluorescently-conjugated monoclonal antibodies: CD8-QDOT 605 and CD4-PE-Texas Red (Invitrogen, Grand Island, NY, USA); CD3-Pacific Blue, CCR5-PE-Cy5, CD38-PE, HLA-DR-FITC, PD-1 Alexa Fluor 647, CD45RA-PE-Cy7 (BD Biosciences, San Jose, CA, USA); and CCR7-APCeFluor 780 (eBioscience, San Diego, CA, USA). Naïve and memory T cell subsets were defined by quadrant gating with FMO controls on a CD45RA versus CCR7 plot.
For cytokine staining, PBMCs were stimulated for 18–22 h at 37°C with overlapping peptide pools corresponding to HIV-1 Con B Gag peptides (NIH 8117) in the presence of 0.5 µg/mL Brefeldin A and 05 µg/mL Monensin (Sigma-Aldrich, St. Louis, MO, USA). A control well with no stimulation was run in parallel for each sample. Cells were washed and stained with AARD, fixed, and permeabilized for intracellular staining with antibodies against CD3-Pacific Blue, IFNγ -FITC, IL-2-PE (BD BioSciences), CD4-PE Texas Red, and CD8-QDOT 605 (Invitrogen). Data were compensated and analyzed using FlowJo V9 (TreeStar, Ashland, OR, USA).
For cytokine staining, PBMCs were stimulated for 18–22 h at 37°C with overlapping peptide pools corresponding to HIV-1 Con B Gag peptides (NIH 8117) in the presence of 0.5 µg/mL Brefeldin A and 05 µg/mL Monensin (Sigma-Aldrich, St. Louis, MO, USA). A control well with no stimulation was run in parallel for each sample. Cells were washed and stained with AARD, fixed, and permeabilized for intracellular staining with antibodies against CD3-Pacific Blue, IFN
8
Multiparametric T cell Characterization
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T cells were washed in staining buffer (SB) consisting of phosphate-buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyd. For intracellular staining, T cells were stimulated for 6 h or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 1:2 and in the presence of BD GolgiPlug and BD Golgistop at a 1/1,000 dilution. Cells were stained both extracellular and intracellular using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc., USA). The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFNγ-FITC, IL-2-APC, TNF-α-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26–35)-specific TCR was detected with dextramer staining (Immudex) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).
9
Cytokine Profiling of T Cell Responses
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Heparinized whole blood (100 μl) was cultured in 96 U bottom culture plate and incubated with PMA/Ionomycin (10 ng/ml and 100 µg/ml, Sigma-Aldrich, UK), P. falciparum 3D7 schizonts lysate (5 µg/ml) and mycobacterium purified protein derivative (10 µg/ml, Statens Serum Institut, Denmark) in parallel with a non-stimulated background control for 24 h. Harvested cells were stained with surface markersCD3-APC-H7, CD4-PerCP (all from BD Biosciences, San Jose, CA, USA), and CD8-Pacific Orange (Invitrogen™ Life technologies, Carlsbad, CA, USA) and lysed as above. The cells were the permeabilized with 500 μl of 1× BD Cytofix/Cytoperm (BD Cytofix/Cytoperm™, San Diego, California) and then stained with intracellular markers IFN-γ-FITC (BD FastImmune™, San Jose, CA, USA) and TNF-PE (BD Biosciences, San Jose, California) for 30 min. The cells were then acquired as above and minimum of 10,000 CD4+ T cell events were collected per sample. Single stained BD comp beads were processed and acquired in parallel to samples each day and used for compensation. Analysis was conducted using FlowJo software version 9.7.5 (Tree Star Inc., San Carlos, CA, USA).
10
MHC Tetramer-Based T-Cell Immunophenotyping
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MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.
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