For TNF-α surface staining, PCMCs were stimulated with 1 μM ionomycin/10 nM PMA or 100 ng/mL LPS at 37°C for 4 hours in the presence of TAPI-1, washed with ice-cold PBS, and incubated at 4°C with Fcblock (Miltenyi Biotec) followed by fluorochrome-conjugated CD117, FcεRI, and TNF-α antibodies diluted in PBS-1% BSA. Intracellular staining of cytokines, IRAP, and VAMP3 was performed using the BD intracellular staining kit and suitable species-specific fluorescent secondary antibodies (Life Technologies). For degranulation experiments, PCMCs were stimulated with 1 μM ionomycin/10 nM PMA or compound 48/80 for 30 minutes at 37°C, placed on ice, and surface stained with Alexa Fluor 488 anti-LAMP1. BD Canto and Gallios flow cytometers were used for cell analysis.
Suitable species specific fluorescent secondary antibodies
Manufactured by Thermo Fisher Scientific
Suitable species-specific fluorescent secondary antibodies are designed to detect and label target proteins or molecules in various biological samples. These antibodies are conjugated with fluorescent dyes that emit light upon excitation, enabling visualization and quantification of the target analytes using fluorescence-based techniques such as flow cytometry, immunofluorescence, and western blotting.
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Lab products found in correlation
2 protocols using suitable species specific fluorescent secondary antibodies
1
Mast Cell Surface Protein Profiling
2
Multiparametric Flow Cytometry of Activated Mast Cells
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For TNF-α surface staining, PCMCs were stimulated with 1µM ionomycin/10nM PMA or 100ng/ml LPS at 37°C for 4h in the presence of TAPI-1, washed with ice-cold PBS, and incubated at 4°C with Fcblock (Miltenyi) followed by fluorochrome-conjugated CD117, FcεRI and TNF-α antibodies diluted in PBS-1% BSA. Intracellular staining of cytokines, IRAP and VAMP3 was performed using the BD intracellular staining kit and suitable species-specific fluorescent secondary antibodies (Life technologies).
For degranulation experiments, PCMCs were stimulated with 1µM ionomycin/10nM PMA or 48/80 for 30 min at 37°C, placed on ice and surface-stained with AlexaFluor488 anti-LAMP1.
BD Canto TM and Gallios flow cytometers were used for cell analysis.
For degranulation experiments, PCMCs were stimulated with 1µM ionomycin/10nM PMA or 48/80 for 30 min at 37°C, placed on ice and surface-stained with AlexaFluor488 anti-LAMP1.
BD Canto TM and Gallios flow cytometers were used for cell analysis.
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