Mice were sacrificed and single cell suspensions were isolated from the spleens. Then, FACS analysis was performed to determine T cell cytokine production, as described previously (33 (link)). The following antibodies were ordered from eBiosciences (San Diego, CA, USA): CY5-conjugated anti-CD4, CY5-conjugated anti-CD8, FITC-conjugated anti-IFN-γ, and FITC-conjugated anti-IL-4.
Fitc conjugated anti ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States
FITC-conjugated anti-IFN-γ is a fluorescently labeled antibody that binds to the cytokine interferon-gamma (IFN-γ). This product is designed for use in flow cytometry and other immunoassay applications to detect and quantify IFN-γ expression.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (1 mentions)
Fix perm kit, by Thermo Fisher Scientific (1 mentions)
Facs arial 2, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Pe conjugated anti cd3, by Thermo Fisher Scientific (1 mentions)
Phorbol 2 myristate 13 acetate, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Roche (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
Apc conjugated anti il 17, by BioLegend (1 mentions)
Fixation permeabilization solution kit, by Thermo Fisher Scientific (1 mentions)
Monesin, by Merck Group (1 mentions)
Pecy7 conjugated anti t bet, by Thermo Fisher Scientific (1 mentions)
Cytomic fc500, by Beckman Coulter (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Facs flow cytometer, by BD (1 mentions)
7 protocols using fitc conjugated anti ifn γ
1
T Cell Cytokine Profiling in Mice
2
Quantification of T-cell Subsets in Spinal Cord
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On day 15, spinal cord tissue was extracted from euthanized mice and pooled for each treatment group (N = 6). Tissue was dissociated and lymphocytes collected at the interface of a 63% - 27% Percoll gradient, plated in Iscove’s Modified Dulbecco’s Medium (IMDM) and stimulated overnight with phorbol 12-myristate 13-acetate (20 nM and ionomycin (1 μM) in IMDM at 37°C. Brefeldin-A was added at a final concentration of 9 μg/ml and incubated for 6 hours. Fc receptors were blocked with 2.4 g2 Fc blocking reagent in 10% normal mouse serum, followed by extracellular staining with anti-CD4+ antibody conjugated to PE. Cells were then washed, fixed, and permeabilized (Fix & Perm kit, Invitrogen, Grand Island, NY) and stained with antibodies to Th1, Th2, and Th17 using FITC conjugated anti-IFN- γ, APC conjugated anti-IL-2, and PerCP Cy5.5 conjugated anti-IL-17, respectively (eBioscience, San Diego, CA) following modifications of published and manufacturer’s instructions [5 (link),6 (link),43 (link)]. Replicate samples of stained cells were processed in the Core Faculty using a FACSCalibur flow cytometer (BD Bioscience).
3
Cytokine Profiling of RA T Cells
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RA patient T cells were co-cultured with RASF for 5 days as described above. At the end of co-culture, the cells were stimulated with PMA (50 ng/ml) plus ionomycin (1000 ng/ml) for 5 h in the presence of Brefeldin A (10 µg/ml). Then, the cells were stained with PerCP-CY5.5–conjugated anti-CD4, fixed and permeabilized, followed by intracellular staining using FITC-conjugated anti-IFN-γ, and Alexa Fluor 647-conjugated anti-IL-17 (eBioscience, San Diego, CA, USA). Percentage of positive cells was analyzed on a FACS Arial II flow cytometer (Becton Dickinson, San Diego, CA, USA).
4
Analyzing Immune Cell Subsets in MLN
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Single-cell suspensions from the MLN were prepared and enriched by nylon wool. Referring to previous studies [25 (link),26 ], we calculated the frequency of CD4+ CD25+FOXP3+ Treg cells in individual samples, as well as that of the effector T cells Th1 (CD3+CD8–interferon [IFN]-γ+), Th2 (CD3+CD8–IL-4+), Th9 (CD3+CD8–IL-9+), Th17 (CD3+CD8–IL-17A+), and Th22 (CD3+CD8–IL-22+IL-17A–). Cells were stained in PBS containing 1% fetal calf serum with the following antibodies (all from eBioscience): phycoerythrin (PE)-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-CD25, and PE-Cy5–conjugated anti-FOXP3; PE-conjugated anti-CD3, allophycocyanin-conjugated anti-CD8, FITC-conjugated anti–IFN-γ, anti–IL-4, anti–IL-9, anti–IL-17A, and PerCP-eFluor–conjugated anti–IL-22. Homotype-independent antibody was used as the negative control. IFN-γ, IL-4, IL-9, IL-17A, and IL-22 intracellular staining was performed after 4-h stimulation with 25 ng/mL phorbol 2-myristate 13-acetate, 1 mg/mL ionomycin, and 2 nmol/mL monensin (all from Sigma-Aldrich).
5
Characterizing Conjunctival Immune Cells
6
Th1 Cell Characterization in Mice
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Cells were collected and stimulated with PMA (25 ng/mL) and ionomycin (1 μg/mL) in the presence of 1 μg/mL monesin (Sigma-Aldrich) for 4 hours. Cells were then fixed, permeabilized by using Fixation/Permeabilization Solution Kit (eBioscience) according to the manufacturer's instructions, and stained with FITC-conjugated anti-IFN-γ, PE-conjugated anti-phospho-STAT4, or PE-cy7-conjugated anti-T-bet (eBioscience). In order to stain Th1 cells in mice, single cell suspension was deprived from spleen of WT and Gnaq−/− chimeric mice, stimulated with PMA, ionomycin, and monensin for 4 hour. After culture, cells were stained with PE-conjugated anti-CD4, followed by intracellular staining with FITC-conjugated anti-IFN-γ. Cells were analyzed on Cytomic FC500 (Beckman Coulter), and data were analyzed using FlowJo (Tree Star).
7
Tumor Immune Profile Characterization
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Tumor masses were minced and digested with a mixture of 0.3 mg/ml DNase I (Sigma-Aldrich) and 0.25 mg/ml Liberase TL (Roche) in serum-free RPMI at 37°C for 25 min. Then the digested pieces were gently pressed between the frosted edges of two sterile glass slides, and the cell suspension was filtered through a 40 μm cell strainer (BD Biosciences). The immune subsets in the tumors were determined by flow cytometry and data were acquired using a FACS flow cytometer (BD Biosciences, San Jose, CA). For intracellular IFN-γ staining, harvested cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 4 h and incubated for the last 1 h with brefeldin A (10 μg/ml). Cells were subjected to intracellular cytokine analysis with anti-IFN-γ antibody. The antibodies used for FACS include APC-conjugated anti-CD45, Pacific blue-conjugated anti-CD8, FITC-conjugated anti-IFN-γ, Alex Flour 488-conjugated anti-p-S6, PerCP/Cy5.5-conjugated anti-CD4, PE-conjugated anti-NK1.1, FITC-conjugated anti-TCRγδ, PE-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Gr-1, and PE-conjugated anti-MHC-II (eBioscience). Data were analyzed by FlowJo software (TreeStar Inc., San Carlos, CA).
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