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Found 3 matching solutions for this experiment
| Upstream tips |
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cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA). |
For flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA). |
| Protocol tips |
| cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA). |
| Downstream tips |
| For flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA). |
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| BM cells were suspended at 1×106/ml in RPMI 1640 with 10% FBS, 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4. Cells were plated into wells of six-well tissue culture plates, and cultured for 6 days |
Ab incubation 30 min at 4°C. After washing, DCs were resuspended in PBS containing 1% BSA and 0.1% NaN3, fixed with 1% paraformaldehyde, and analyzed by a FACScan flow cytometer (BD Immunocytometry Systems, Mountain View, CA) |
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| Upstream tips |
| BM cells were suspended at 1×106/ml in RPMI 1640 with 10% FBS, 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4. Cells were plated into wells of six-well tissue culture plates, and cultured for 6 days |
| Protocol tips |
| Ab incubation 30 min at 4°C. After washing, DCs were resuspended in PBS containing 1% BSA and 0.1% NaN3, fixed with 1% paraformaldehyde, and analyzed by a FACScan flow cytometer (BD Immunocytometry Systems, Mountain View, CA) |
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Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
Cells were acquired on the BD FACSCANTO II and LSR (BD Biosciences) and analysis was carried out using FlowJo (Tree Star). |
| Protocol tips |
| Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
| Downstream tips |
| Cells were acquired on the BD FACSCANTO II and LSR (BD Biosciences) and analysis was carried out using FlowJo (Tree Star). |
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