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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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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. 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 |
| 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 |
| Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours. |
For blocking of nonspecific Fc-mediated interactions splenocytes were pre-incubated with 1 μg of anti-mouse CD16/CD32 for 5 minutes. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry. |
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| Upstream tips |
| Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours. |
| Protocol tips |
| For blocking of nonspecific Fc-mediated interactions splenocytes were pre-incubated with 1 μg of anti-mouse CD16/CD32 for 5 minutes. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry. |
| Upstream tips |
Protocol tips |
Downstream tips |
| Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours. |
For blocking of nonspecific Fc-mediated interactions splenocytes were pre-incubated with 1 μg of anti-mouse CD16/CD32 for 5 minutes. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry. |
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| Upstream tips |
| Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours. |
| Protocol tips |
| For blocking of nonspecific Fc-mediated interactions splenocytes were pre-incubated with 1 μg of anti-mouse CD16/CD32 for 5 minutes. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry. |
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