APC-Cy™7 Mouse Anti-Human CD3

Protocol tips

Upstream tips	Protocol tips	Downstream tips
FACS staining was done in FACS buffer (PBS, 1% BSA). For measuring intracellular cytokines, PBMCs were pre-treated for 15 min with the hydroxamic acid derivative GM6001 (100 μM, CALBIOCHEM) to inhibit the shedding of IL-6Rα (4) and stimulated for 4 hours with or without phorbol myristate acetate (50ng/ml, PMA) and ionomycin (1 μg/ml) (both from Sigma Aldrich) in the presence of Golgiplug (BD Bioscience)	For intracellular staining, stained cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience).	Cells were analyzed on an LSRII® flow cytometer. In most cases, greater than 100,000 events were collected for analysis. Collected data were analyzed using FlowJo software (Tree Star).

Upstream tips
FACS staining was done in FACS buffer (PBS, 1% BSA). For measuring intracellular cytokines, PBMCs were pre-treated for 15 min with the hydroxamic acid derivative GM6001 (100 μM, CALBIOCHEM) to inhibit the shedding of IL-6Rα (4) and stimulated for 4 hours with or without phorbol myristate acetate (50ng/ml, PMA) and ionomycin (1 μg/ml) (both from Sigma Aldrich) in the presence of Golgiplug (BD Bioscience)
Protocol tips
For intracellular staining, stained cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience).
Downstream tips
Cells were analyzed on an LSRII® flow cytometer. In most cases, greater than 100,000 events were collected for analysis. Collected data were analyzed using FlowJo software (Tree Star).

Publication protocol

Following mouse antibodies were used for flow cytometric analysis: APC-Cy7-CD3, Pacificblue-CD8, PE-Cy5-CD45RA, PE-Cy7-CCR7, PE-gp130, PE-perforin, Alexa Fluor 700-granzyme B, APC or APC-Cy7-IFN-γ, APC-IL-5, BV711 or APC-IL-13, APC- or PE-IL-6Rα, APC-Cy7-IL-2, PE-2B4, PE-CCR4, PE-CCR5, PE-CCR6, PE-T-bet and Alexa Fluor 647-Eomes (all from BD Bioscience except the latter eight antibodies from BioLegend). Also, goat anti-human IL-7Rα (R&D Systems), FITC-donkey anti-goat IgG (Santa Cruz Biotechnology) and mouse PE-ICAM-1 antibodies (ebioscience) were used. FACS staining was done in FACS buffer (PBS, 1% BSA). For measuring intracellular cytokines, PBMCs were pre-treated for 15 min with the hydroxamic acid derivative GM6001 (100 μM, CALBIOCHEM) to inhibit the shedding of IL-6Rα (4) and stimulated for 4 hours with or without phorbol myristate acetate (50ng/ml, PMA) and ionomycin (1 μg/ml) (both from Sigma Aldrich) in the presence of Golgiplug (BD Bioscience) (5). For intracellular staining, stained cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience). Cells were analyzed on an LSRII® flow cytometer. In most cases, greater than 100,000 events were collected for analysis. Collected data were analyzed using FlowJo software (Tree Star).

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Manufacturer protocol

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