Brilliant Violet 605™ anti-human CD69 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Cells were stained with a fixable Live/Dead dye (ThermoFisher Scientific) concurrently with saturating concentrations of surface mAbs (Table S2) in a buffer containing 50%ABrilliant Violet Buffer (BD Bioscience) and 50%APBS for 30min at 4°C.	Intracellular proteins were detected with saturating concentrations of mAbs for 30mins at 4°C in either a 0.1% saponin (SigmaAAldrich) buffer containing 10% FBS (SigmaAAldrich) or 1x PBS. All samples were acquired in a 0.1% saponin buffer on an X20FortessaASORP (BD Biosciences) and analysed in FlowJo (TreeStar).

Upstream tips
Cells were stained with a fixable Live/Dead dye (ThermoFisher Scientific) concurrently with saturating concentrations of surface mAbs (Table S2) in a buffer containing 50%ABrilliant Violet Buffer (BD Bioscience) and 50%APBS for 30min at 4°C.
Protocol tips
Intracellular proteins were detected with saturating concentrations of mAbs for 30mins at 4°C in either a 0.1% saponin (SigmaAAldrich) buffer containing 10% FBS (SigmaAAldrich) or 1x PBS. All samples were acquired in a 0.1% saponin buffer on an X20FortessaASORP (BD Biosciences) and analysed in FlowJo (TreeStar).

Publication protocol

Cells were stained with a fixable Live/Dead dye (ThermoFisher Scientific) concurrently with saturating concentrations of surface mAbs (Table S2) in a buffer containing 50%ABrilliant Violet Buffer (BD Bioscience) and 50%APBS for 30min at 4°C. Once stained PBMC and IHL were fixed and permeabilised, where necessary, with either Cytofix/Cytoperm (BD Bioscience), or with the FoxP3 Buffer Kit (BD Bioscience) according to manufacturer’s instructions. Intracellular proteins were detected with saturating concentrations of mAbs for 30mins at 4°C in either a 0.1% saponin (SigmaAAldrich) buffer containing 10% FBS (SigmaAAldrich) or 1x PBS. All samples were acquired in a 0.1% saponin buffer on an X20FortessaASORP (BD Biosciences) and analysed in FlowJo (TreeStar).

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Manufacturer protocol

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