Single cell suspensions were prepared from the indicated tissues as previously reported6 (link). Data were acquired on LSR Fortessa or LSRII flow cytometers (BD Biosciences) and analyzed using FlowJo v9.3 (FlowJo) or software designed by the Division of Computer Research and Technology, NCI. Live cells were gated using forward scatter exclusion of dead cells stained with propidium iodide. For fixed cells, dead cells were excluded by Aqua Live/Dead (Invitrogen) staining and fixation and permeabilization were performed with IC fixation and permeabilization buffers (eBioscience). Cytokine expression was assessed on cells upon stimulated in vitro with PMA (25 ng/ml) and ionomycin (1 μM) for 4 hours as previously described53 (link).
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Single Cell Suspension Preparation and Cytokine Analysis
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Corresponding Organization : Seoul National University Dental Hospital
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Variable analysis
independent variables
- Stimulation of cells with PMA (25 ng/ml) and ionomycin (1 μM) for 4 hours
- Cytokine expression
- Live cells gated using forward scatter exclusion of dead cells stained with propidium iodide
- For fixed cells, dead cells excluded by Aqua Live/Dead (Invitrogen) staining
- Fixation and permeabilization performed with IC fixation and permeabilization buffers (eBioscience)
- Not explicitly mentioned
- Not explicitly mentioned
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