For secondary outcomes 1 to 4, peripheral blood mononuclear cells will be isolated and subjected to flow cytometric analysis as described previously by our group.[29 ] The monoclonal antibodies will be anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, anti-IFN-γ-PE-Cy7, anti-TNF-α-APC, anti-IL-17A-APC, anti-granzyme B-PE, and anti-perforin-APC (all from eBioscience, San Diego, CA, USA). Permeabilized cells will be stained for intracellular cytokines and cytotoxic molecules using anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry will employ a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and the data will be analyzed with aid of FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA).
Anti ifn γ pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-IFN-γ-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the interferon-gamma (IFN-γ) protein. It is designed for use in flow cytometry applications to detect and quantify IFN-γ-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by Thermo Fisher Scientific (6 mentions)
Anti il 4 pe, by Thermo Fisher Scientific (5 mentions)
Anti cd4 af700, by Thermo Fisher Scientific (4 mentions)
Anti cd25 apc, by Thermo Fisher Scientific (4 mentions)
Anti foxp3 pe cy7, by Thermo Fisher Scientific (4 mentions)
Canto software, by BD (4 mentions)
Facscanto 2 cytometer, by BD (4 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (4 mentions)
Anti cd4 ecd, by Beckman Coulter (4 mentions)
Anti cd3 alexa fluor 700, by Exbio (4 mentions)
Anti cd8 hv500, by BD (4 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (4 mentions)
Brefeldin a, by BioLegend (4 mentions)
Anti cd8 apc, by Thermo Fisher Scientific (4 mentions)
Anti cd8 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd28 apc, by Thermo Fisher Scientific (3 mentions)
Anti cd57 fitc, by Thermo Fisher Scientific (3 mentions)
Anti tnf α apc, by Thermo Fisher Scientific (3 mentions)
Anti il 17a apc, by Thermo Fisher Scientific (3 mentions)
Anti granzyme b pe, by Thermo Fisher Scientific (3 mentions)
28 protocols using anti ifn γ pe cy7
1
Multicolor Flow Cytometry Analysis of PBMC
2
Flow Cytometric Analysis of Cytokine Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated splenocytes (1 × 106 cells/well) were stimulated for 1 h with 5 µg/ml MOMP in the presence of 1 µg/ml anti-CD28 and anti-CD49d mAbs (both BD Pharmingen, San Diego, CA, USA) in a total volume of 200 µl supplemented with RPMI 1640. The cells were subsequently incubated for 5 h at 37℃ after addition of 10 µg/ml brefeldin A (Sigma Aldrich) and 0.7 µl/ml monensin/Golgi-stop (BD Pharmingen). Following overnight storage at 4℃, the cells were washed in FACS buffer and stained with anti-CD4 (APC) and anti-CD44 (FITC) mAbs (BD Biosciences) in FACS buffer for 30 min at 4℃. Cells were washed with FACS buffer, permeabilized by using the Cyto-fix/Cyto-perm kit (BD Pharmingen) according to the manufacturer’s instructions, and stained intracellularly for 30 min at 4℃ using anti-IFN-γ (PE–Cy7) and anti-IL-17a (PerCP–Cy5.5) mAbs (eBiosciences, San Diego, CA, USA) in Perm wash buffer. After washing, the cells were re-suspended in FACS buffer and analysed by flow cytometry using a six-colour BD FACS Canto flow cytometer (BD Biosciences). Responses were analysed with the FlowJo software (Tree Star Inc., Ashland, OR, USA).
3
Evaluating T-cell Subsets in Asthmatic Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the recruitment of Th1, Th2, and Tregs induced by ASCs treatment, LLN cells from OVA-induced asthmatic mice and ASCs-treated asthmatic mice were cultured in anti-CD3-coated plate for 6 h. To evaluate To evaluate CD4+CD25+Foxp3+ (Tregs) and IL-10+/CD4+ T cells, cells were stained with anti-CD4-FITC (0.5 mg/ml) and anti-CD25-APC (0.2 mg/ml) in accordance with the manufacturer’s recommendations (eBiosciences, San Diego, CA). After surface staining, the cells were permeabilized using a Cytofix/Cytoperm Kit (eBiosciences). After permeabilization, the cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBiosciences).
To assess the Th1 and Th2 cell population, LLNs cells were stained with an anti-CD4-FITC Ab. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
To assess the Th1 and Th2 cell population, LLNs cells were stained with an anti-CD4-FITC Ab. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
4
Analyzing T Cell Subsets in Asthma Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the recruitment of Th1, Th2, and Treg induced by ASC sup treatment, the LLN cells of the OVA-induced animal model of acute asthma and ASC sup-treated animal model of acute asthma were cultured on anti-CD3-coated plates for 6 hours. To determine the CD4+CD25+Foxp3+ (Treg) and IL-10+/CD4+ T cell populations, the cells were stained with anti-CD4-FITC (0.5 mg/ml) and/or anti-CD25-APC (0.2 mg/ml) in accordance with the manufacturer’s recommendations (eBioscience, San Diego, CA, USA). After surface staining, the cells were permeabilized using a Cytofix/Cytoperm kit (eBioscience). After permeabilization, the cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBioscience). To quantify the Th1 and Th2 cell populations, the LLN cells were stained with an anti-CD4-FITC antibody. After surface staining, the CD4+ T cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBioscience) and anti-IL-4-PE (eBioscience) antibodies. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
5
Multiparameter Flow Cytometric Analysis of T-cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A single-cell suspension was prepared from blood sample or spleen sample. Blood cells were incubated with anti-CD3-PE and anti-CD19-PE-Cy7 monoclonal antibody (BD Biosciences, USA) at 4°C for 30 min. Spleen cells were incubated with anti-CD3-PE, anti-CD4-FITC, and anti-CD8-Percp-Cy5.5 monoclonal antibody (BD Biosciences, USA) at 4°C for 30 min. For intracellular staining, cells were fixed and permeabilized after surface staining, and anti-IL-4-APC and anti-IFN-γ-PE-Cy7 (eBioscience, USA) were then added to stain Th2 cells and Th1 cells. Stained cells were analyzed by flow cytometric analysis using a flow cytometer (ACEA NovoCyte 2060R, China).
6
Functional Profiling of HIV-Infected T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The functional profile response of T cells was evaluated by measuring production of cytokines and effector molecules by flow cytometry. Approximately 1x105 of PBCMs treated with calcitriol or EtOH and infected with HIV-1 from Co-HC, were cultured for 24 hours, in presence of brefeldin (1ug/mL) and monensin (1ug/mL). Extracellular staining for CD4+ and CD8+ T cells was done, as previously described, and intracellular staining was performed using the antibodies anti-IL-2-FITC, anti-IFN-γ-PeCy7, anti-TNF-α-PerCp-Cy5.5, anti-MIP-1β-PE, anti-granzyme-FITC and anti-perforin-PE (eBioscience).
7
Functional T Cell Analysis in Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mononuclear cells isolated from fresh tumor specimens were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) + 1 μg/ml of ionomycin for 1 h followed by 3 h incubation with brefeldin A (BioLegend). Unstimulated cells were used as a control. The cells were then washed in PBS, stained with anti-CD3 Alexa Fluor 700 (EXBIO), anti-CD4 ECD (Beckman Coulter) and anti-CD8 HV500 (BD Biosciences), fixed using fixation/permeabilization buffer (eBioscience), permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with an anti-IFN-γ PE-Cy7 (eBioscience), anti-granzyme B Brilliant Violet 421 (BD Biosciences) (Additional file 1 : Table S4). The percentage of CD3+CD8+ T cells producing IFN-γ and degranulating upon PMA/ionomycin stimulation were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.). Gating strategy is depicted in Additional file 1 : Figure S4.
8
T Cell Activation Assay with rF1-V and SA-4-1BBL
9
Evaluating T-cell Subsets in Asthma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the recruitment of Th1, Th2, and Tregs induced by ASCs treatment, LLN cells from OVA-induced asthmatic mice and ASC-treated asthmatic mice were cultured in anti-CD3-coated plates for 6 h. To evaluate CD4+CD25+Foxp3+ (Tregs) and IL-10+/CD4+ T-cells, cells were stained with anti-CD4-FITC (0.5 mg/mL) and anti-CD25-APC (0.2 mg/mL) following the manufacturer’s recommendations (eBiosciences, San Diego, CA). After surface staining, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences). After permeabilization, cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBiosciences).
To assess the Th1 and Th2 cell populations, LLN cells were stained with anti-CD4-FITC Ab. After surface staining, CD4+ T-cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
To assess the Th1 and Th2 cell populations, LLN cells were stained with anti-CD4-FITC Ab. After surface staining, CD4+ T-cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
10
Intracellular Cytokine Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of intracellular cytokine expression T-cells were stimulated with phorbol myristate acetate (PMA), Ionomycin for 1 hour and Brefeldin A for 4.5 hours. All chemicals were obtained from Sigma-Aldrich (Munich, Germany). Cells were harvested and prepared for analysis using the Cytofix/Cytoperm kit (BD Bioscience, Heidelberg, Germany). For intracellular cell staining the following antibodies were used: anti-IL4-FITC, anti-IL17-APC, anti-IFNγ-PECy7, and anti-FOXP3-APC, obtained from eBioscience (San Diego, USA) and anti-Granzyme-PE (BD Bioscience, Heidelberg, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!