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 interleukin il 17a apc
Manufactured by Thermo Fisher Scientific
Sourced in Israel
The Anti-interleukin (IL)-17A-APC is a fluorescently-labeled antibody that specifically binds to the interleukin-17A (IL-17A) cytokine. It can be used in flow cytometry applications to detect and quantify IL-17A-producing cells.
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3 protocols using anti interleukin il 17a apc
1
T cell Cytokine Expression Analysis
2
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.
3
Isolation and Analysis of Intestinal Immune Cells
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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. ( 21) 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) at concentration of 1 million per ml 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 5h, 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)-g-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 5h, 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)-g-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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