Lungs were harvested, digested, and lung single-cell suspensions were restimulated with 50 ng/ml of PMA (Sigma Aldrich), 1 μg/ml of ionomcyin (Sigma Aldrich) and 0.07% Golgi-Stop (BD Biosciences) for 5 hours at 37⁰C in RPMI+10% FBS. Following restimulation, cells were stained with viability dye (UV Ghost dye 450, Tonbo), blocked with an anti-FcR antibody (clone 2.4G2), and surface stained with biotin-labeled anti-CD3 (clone 17A2), PE-Cy5 anti-CD4 (clone 129.19), BV786 anti-CD90.2 (clone 53–2.1), Alexa Fluor 700 anti-CD45 (clone 30-F11), Alexa Fluor 488 anti-CD25 (clone OC61), PE-Cy7 anti-CD127 (clone A7R34) and FITC anti-γδTCR (clone GL3) antibodies, followed by APC-Cy7 streptavidin staining (1:250). Cells were then fixed, permeabolized using the Foxp3/transcription factor staining kit (Tonbo), and intracellularly stained with PE-IL-13 (clone eBio13A), PE-Cy7 anti-IL-17A (clone eBio17B7), and/or PE-IL-4 (clone 11B11). Flow cytometry analysis was conducted on LSRII flow cytometer, and all data were processed using FlowJo software version 10.
Pe cy7 anti il 17a
Manufactured by Cytek Biosciences
The PE-Cy7 anti-IL-17A is a fluorescently-labeled antibody used to detect and quantify the presence of interleukin-17A (IL-17A) in biological samples. It is designed to bind specifically to IL-17A, allowing for its identification and measurement through flow cytometry or other immunoassay techniques.
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2 protocols using pe cy7 anti il 17a
1
Comprehensive Lung Immune Cell Profiling
2
Th17 Cell Differentiation and Analysis
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Three days after Th17 cells differentiation, cells were restimulated with 50 ng/ml PMA, 1 μM ionomycin, and 0.07% of Golgi Stop for 4 h. Following restimulation, cells were stained with viability dye (Ghost Dye UV 450; Tonbo Biosciences), blocked with an anti-FcR Ab (clone 2.4G2), and surface stained with APC-Cy7 anti-CD3 (clone 145-2C11), FITC anti-CD4 (clone GK1.5), BV786 anti-CD90.2 (clone 53-2.1), and APC anti-IL-23R (clone 12B2B64). Cells were then fixed, permeabilized using the Foxp3/transcription factor staining kit (Tonbo Biosciences), and intracellularly stained PE-Cy7 anti–IL-17A (clone eBio17B7). Flow cytometry analysis was conducted on LSR II flow cytometer, and all data were processed using FlowJo software version 10. Antibodies were purchased from Thermo fisher (Invitrogen). In select experiments, Cell Tracer Violet dye was added per manufacturer's instructions (ThermoFisher) prior to Th17 cell differentiation. Th17 cell proliferation was determined by flow cytometry 3 days later by gating on viable, CD4+ IL-17A+ cells and measuring the proliferation index using FlowJo software.
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