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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
| For flow cytometry analysis, BD GolgiPlug Protein Transport Inhibitor (BD Biosciences, San Jose, CA) was added, and cells were cultured for 10 h in 5% CO2 at 37 °C. |
After incubation, cells were washed with FACS buffer (PBS with 2% FBS) and stained with Acqua Live/Dead (Invitrogen, USA) for 10 min at 4 °C for dead cell exclusion. Cells were washed and stained for surface molecules for 30 min at 4 °C, fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm, BD Biosciences, USA). After washing, cells were incubated with antibodies against intracellular antigens for 30 min at 4 °C. Cells were then washed and suspended in 200 μL of FACS buffer for cytometry analysis. |
Data were collected using an LSR II (BD Immunocytometry Systems, USA) with Diva (BD Biosciences, USA). At least 100,000 gated events were acquired. |
| Upstream tips |
| For flow cytometry analysis, BD GolgiPlug Protein Transport Inhibitor (BD Biosciences, San Jose, CA) was added, and cells were cultured for 10 h in 5% CO2 at 37 °C. |
| Protocol tips |
| After incubation, cells were washed with FACS buffer (PBS with 2% FBS) and stained with Acqua Live/Dead (Invitrogen, USA) for 10 min at 4 °C for dead cell exclusion. Cells were washed and stained for surface molecules for 30 min at 4 °C, fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm, BD Biosciences, USA). After washing, cells were incubated with antibodies against intracellular antigens for 30 min at 4 °C. Cells were then washed and suspended in 200 μL of FACS buffer for cytometry analysis. |
| Downstream tips |
| Data were collected using an LSR II (BD Immunocytometry Systems, USA) with Diva (BD Biosciences, USA). At least 100,000 gated events were acquired. |
| Upstream tips |
Protocol tips |
Downstream tips |
| Spleens and tumors were excised from the 4T1 tumor-bearing mice. 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). |
Abs were added to the cells and incubated for 30 min. Cells were then fixed and permeabilized, and intracellular staining was performed using an anti-granzyme B PE antibody (clone NGZB, eBioscience). All staining reactions were performed in a final volume of 100 μl at 4 °C. |
Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA). |
| Upstream tips |
| Spleens and tumors were excised from the 4T1 tumor-bearing mice. 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 |
| Abs were added to the cells and incubated for 30 min. Cells were then fixed and permeabilized, and intracellular staining was performed using an anti-granzyme B PE antibody (clone NGZB, eBioscience). All staining reactions were performed in a final volume of 100 μl at 4 °C. |
| Downstream tips |
| Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA). |
| Upstream tips |
Protocol tips |
Downstream tips |
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IgG isotype controls were used as parallels. At last, we applied ALSRFortessa flow cytometry platform and Flowjo Software to analyze output data. |
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| Protocol tips |
| IgG isotype controls were used as parallels. At last, we applied ALSRFortessa flow cytometry platform and Flowjo Software to analyze output data. |
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