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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream 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). |
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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). |
| Upstream 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. 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). |
| Upstream tips |
Protocol tips |
Downstream tips |
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Fc receptors were blocked with anti-CD16/CD32 for 15 min (5 μg/ml in PBS/BSA, clone 2.4G2, in house production). Subsequently, cells were washed with ice-cold PBS/0.5%BSA, and incubated with fluorescent labeled antibodies for 10 min on ice. After washing twice, cells were re-suspended in PBS/0.5% BSA/2 mM EDTA, and analyzed in an LSRII flow cytometer (BD Biosciences). |
The resulting data were analyzed using the FlowJo software. |
| Protocol tips |
| Fc receptors were blocked with anti-CD16/CD32 for 15 min (5 μg/ml in PBS/BSA, clone 2.4G2, in house production). Subsequently, cells were washed with ice-cold PBS/0.5%BSA, and incubated with fluorescent labeled antibodies for 10 min on ice. After washing twice, cells were re-suspended in PBS/0.5% BSA/2 mM EDTA, and analyzed in an LSRII flow cytometer (BD Biosciences). |
| Downstream tips |
| The resulting data were analyzed using the FlowJo software. |
| Upstream tips |
Protocol tips |
Downstream tips |
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Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell) |
Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences). |
| Protocol tips |
| Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell) |
| Downstream tips |
| Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences). |
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