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Found 3 matching solutions for this experiment
| 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). |
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). |
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| 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 |
| 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 |
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. |
After incubation cells were washed with fluorescence-activated cell sorting buffer and stained with surface antibodies |
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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 |
| After incubation cells were washed with fluorescence-activated cell sorting buffer and stained with surface antibodies |
| Upstream tips |
Protocol tips |
Downstream tips |
| For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS. |
After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature. |
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| Upstream tips |
| For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS. |
| Protocol tips |
| After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature. |
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