LPMC were seeded in 96-well U-bottom culture dishes and stimulated with phorbol myristate acetate (PMA) (10 ng/mL), ionomycin (1 µg/mL), and brefeldinA (10 µg/mL;eBioscience, San Diego, CA). After 5 h, cells were stained with anti-CD3-PerCP (1:50, final dilution, BD Biosciences, San Jose, CA) and fixed with 1% formaldehyde for 20′. Subsequently cells were permeabilized with 0.5% saponin in 1% BSA FACS buffer and stained with the following Abs: anti-interferon (IFN)-γ-PE (1:50, final dilution; BD Biosciences), anti–interleukin (IL)-17A–APC (1:50, final dilution, eBioscience). Appropriate isotype-matched controls from BD Biosciences were included in all of the experiments. Cells were analysed using a FACS Calibur cytometer and Cell-QuestPro software.
Anti interferon ifn γ pe
Manufactured by BD
The Anti-interferon (IFN)-γ-PE is a laboratory reagent used for the detection and analysis of interferon-gamma (IFN-γ) in biological samples. It is a fluorescent-conjugated antibody that binds specifically to IFN-γ, allowing for its quantification and evaluation in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Thermo Fisher Scientific (2 mentions)
Anti cd3 percp, by BD (2 mentions)
Anti interleukin il 17a apc, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Anti il 4 pe, by BD (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
Anti il 17 pe, by BD (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Anti cd3 apc, by BD (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
3 protocols using anti interferon ifn γ pe
1
T cell Cytokine Expression Analysis
2
Intracellular Cytokine Profiling of Immunized Mice
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For the intracellular cytokine staining assay, the mice were immunized with three doses (10, 100 and 1000 ng) of K. pneumoniae EVs using the above protocol. A week after the last immunization, the spleens of both immunized and non-immunized mice were extracted for use in the re-stimulation assay. The separated splenocytes were incubated in 48-well plates coated with 1 μg ml−1 of anti-CD3 (eBioscience) and anti-CD28 (eBioscience) antibodies at 37 °C for 12 h. The splenocytes were stained with anti-CD3-APC (BD Biosciences) and anti-CD4-FITC (BD Biosciences) antibodies for 30 min at 4 °C followed by fixation for 10 min in 4% paraformaldehyde at room temperature. The splenocytes were then incubated with antibodies against the cytokines of interest (anti-interferon (IFN)-γ-PE, anti-IL-17-PE and anti-IL-4-PE; BD Biosciences) for 30 min at room temperature followed by analysis using a FACSCalibur flow cytometer (BD Biosciences) with the CELLQuest software.34 (link)
3
Cytokine Expression in Inflammatory Bowel Disease
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Lamina propria mononuclear cells (LPMC) were isolated from ileal biopsy samples and intestinal resection specimens of CD patients and CTR as described elsewhere. (20) LPMC were suspended in RPMI 1640 medium, supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (P) (100 U/ml), and streptomycin (S) (100 µg/ml) (Life Technologies-GibcoCRL, Milan, Italy) and used to assess cytokine expression by ow cytometry.
Flow-cytometry analysis LPMC were seeded in 96-well U-bottom culture dishes and stimulated with phorbol myristate acetate (PMA) (10 ng/mL), ionomycin (1 µg/mL), and brefeldinA (10 µg/mL;eBioscience, San Diego, CA). After 5 h, cells were stained with anti-CD3-PerCP (1:50, nal dilution, BD Biosciences, San Jose, CA) and xed with 1% formaldehyde for 20'. Subsequently cells were permeabilized with 0.5% saponin in 1% BSA FACS buffer and stained with the following Abs: anti-interferon (IFN)-γ-PE (1:50, nal dilution; BD Biosciences), anti-interleukin (IL)-17A-APC (1:50, nal dilution, eBioscience). Appropriate isotype-matched controls from BD Biosciences were included in all of the experiments. Cells were analysed using a FACS Calibur cytometer and Cell-QuestPro software.
Flow-cytometry analysis LPMC were seeded in 96-well U-bottom culture dishes and stimulated with phorbol myristate acetate (PMA) (10 ng/mL), ionomycin (1 µg/mL), and brefeldinA (10 µg/mL;eBioscience, San Diego, CA). After 5 h, cells were stained with anti-CD3-PerCP (1:50, nal dilution, BD Biosciences, San Jose, CA) and xed with 1% formaldehyde for 20'. Subsequently cells were permeabilized with 0.5% saponin in 1% BSA FACS buffer and stained with the following Abs: anti-interferon (IFN)-γ-PE (1:50, nal dilution; BD Biosciences), anti-interleukin (IL)-17A-APC (1:50, nal dilution, eBioscience). Appropriate isotype-matched controls from BD Biosciences were included in all of the experiments. Cells were analysed using a FACS Calibur cytometer and Cell-QuestPro software.
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