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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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The cells were counted and 1×106 cells were used for surface staining. For intracellular staining 1×106 cells were cultured per well in 24 well plates (Nunc, USA) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 750 ng/ml ionomycin (Sigma, USA) overnight, and 10 µg/ml brefeldin A (eBiosciences, USA) was added during the last 4 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers |
After staining, cells were washed again with PBS and cells were fixed with 100 µl fixation buffer (eBiociences, USA) for 30 minutes, then re-suspended in 200 µl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto™ II, BD Biosciences, USA). |
| Protocol tips |
| The cells were counted and 1×106 cells were used for surface staining. For intracellular staining 1×106 cells were cultured per well in 24 well plates (Nunc, USA) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 750 ng/ml ionomycin (Sigma, USA) overnight, and 10 µg/ml brefeldin A (eBiosciences, USA) was added during the last 4 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers |
| Downstream tips |
| After staining, cells were washed again with PBS and cells were fixed with 100 µl fixation buffer (eBiociences, USA) for 30 minutes, then re-suspended in 200 µl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto™ II, BD Biosciences, USA). |
| Upstream tips |
Protocol tips |
Downstream tips |
| the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL. |
The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES. |
The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter). |
| Upstream tips |
| the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL. |
| Protocol tips |
| The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES. |
| Downstream tips |
| The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter). |
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7AAD (Biolegend) or Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
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| Protocol tips |
| 7AAD (Biolegend) or Zombie Aqua Fixable Viability Kit (Biolegend) was used to exclude dead cells. For intracellular transcription factor staining, surface-stained cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer's protocol |
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