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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). |
When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Protocol tips |
| Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). |
| Downstream tips |
| When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| 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). |
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| 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). |
| 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). |
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| 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). |
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