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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies) |
| Protocol tips |
| For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
| Downstream tips |
| All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies) |
| Upstream tips |
Protocol tips |
Downstream tips |
|
For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies) |
| Protocol tips |
| For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C. |
| Downstream tips |
| All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies) |
| Upstream tips |
Protocol tips |
Downstream tips |
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cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. |
After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software. |
| Protocol tips |
| cells were transferred, centrifugation at 1500 g for 10 min and incubated with antibodies for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. |
| Downstream tips |
| After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software. |
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