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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 |
| Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England). |
Anti-CD4 FITC (clone H129.19, BD Biosciences) used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added. |
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| Upstream tips |
| Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England). |
| Protocol tips |
| Anti-CD4 FITC (clone H129.19, BD Biosciences) used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added. |
| Upstream tips |
Protocol tips |
Downstream tips |
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Fc receptors were blocked, 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, 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. |
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