PE-Cy™7 Rat Anti-Mouse TNF

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.	Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences).

Protocol tips
Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.
Downstream tips
Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences).

Publication protocol

"Intracellular cytokine staining was performed as follows: Liver lymphocytes were adjusted to ~5×106 cells/ml in RPMI 1640 culture medium supplemented with 10% FCS and stimulated with 100 ng/ml phorbol myristate acetate plus 1 µg/ml ionomycin at 37°C for 4 h in the presence of the secretion inhibitor monensin (0.16 µg/ml; BD Biosciences) (29). Cells were blocked using 0.5 µg anti-CD16/32 antibody for 10 min at 4°C and then stained with anti-TCR γδ mAb for 30 min at 4°C, followed by washing with PBS and fixing in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma-Aldrich; Merck KGaA) and incubated with anti-IFN-γ and anti-TNF-α for 30 min at 4°C.

Stained cells were immediately analyzed using the FACSCanto™ II flow cytometer (BD Immunocytometry Systems; BD Biosciences). Data were analyzed using FACSDiva™ 2.0 software (BD Immunocytometry Systems; BD Biosciences). Cell gating strategies were as follows: The population of cells double positive for γδ TCR and CD3 was defined as the γδ T cell subtype, which was subsequently subdivided into several subsets, including CD25+, CD69+, IFN-γ+ or TNF-α+ γδ T cells, according to their positivity in the FACS dot plots. CD3 and NK1.1 were used to measure the presence of mouse NK and NKT cells; CD3- NK1.1+ cells were defined as NK cells (left upper quadrant) and CD3+ NK1.1+ cells were defined as NKT cells (right upper quadrant) (30)."

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