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Found 3 matching solutions for this experiment
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For all experiments, 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. |
| Protocol tips |
| For all experiments, 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. |
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DAPI− single cells were evaluated for all analyses except for intracellular stains. To purify mononuclear phagocyte populations, BM was sorted with an InFlux cell sorter (BD) to achieve >97% purity. |
Multiparameter analyses of stained cell suspensions were performed on an LSRII (BD) and analyzed with FlowJo software (Tree Star, Inc.). |
| Protocol tips |
| DAPI− single cells were evaluated for all analyses except for intracellular stains. To purify mononuclear phagocyte populations, BM was sorted with an InFlux cell sorter (BD) to achieve >97% purity. |
| Downstream tips |
| Multiparameter analyses of stained cell suspensions were performed on an LSRII (BD) and analyzed with FlowJo software (Tree Star, Inc.). |
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| To harvest them, culture supernatants were removed and cells were incubated with 2 mM EDTA for 10 min, and then harvested with cell scrapers. |
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| Upstream tips |
| To harvest them, culture supernatants were removed and cells were incubated with 2 mM EDTA for 10 min, and then harvested with cell scrapers. |
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