Protocol tips
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Protocol tips |
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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). |
Protocol 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. |
Downstream 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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