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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences. |
). A total of 200,000 events were acquired for assessment of these investigated antibodies.
Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A). |
| Protocol tips |
| After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin. CD30‐PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences. |
| Downstream tips |
). A total of 200,000 events were acquired for assessment of these investigated antibodies.
Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non‐viable cells, debris, and aggregates were excluded based on forward scatter‐height/forward scatter‐area (FSC‐H/FSC‐A). |
| 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. |
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). |
| 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 |
| 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. |
| Downstream tips |
| 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 |
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100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. |
The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired. |
| Protocol tips |
| 100 µL of the test sample (fresh bone marrow) was added in all tubes. The cells were incubated for 20 min at room temperature (18–25°C) in dark. The RBCs were lysed using 2 ml of 1× BD FACS lysing solution (BD Biosciences, San Jose, CA, USA). The above tubes were centrifuged for 5 min at 1800 g at room temperature. Supernatant was discarded and the pellet was re-suspended. The pellet was washed twice with 2 ml of sheath fluid (FACSFlow, Becton Biosciences, San Jose, CA, USA) at 1800 g for 5 min. |
| Downstream tips |
| The pellet was obtained after washing and was resuspended in 300 µl of sheath fluid in the tube and a total of 20,000 events were acquired. |
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