Whole skin cells were prepared from mouse back skin 4 (link). In brief, skin tissues were digested with a buffer containing collagenase IV, hyaluronidase, and DNase I. Skin cells were labeled with CFSE and stimulated with rIL- 23 (1ng/ml, eBioscience), rIL-1β (1ng/ml, eBioscience), or rIL-23 plus rIL-1β for 3 days. Cell proliferation and intracellular IL-17 were measured by flow cytometry. For blocking experiment, neutralizing mAbs for IL-1β (2μg/ml, eBioscience), IL-23 (12.5μg/ml, eBioscience), or matched isotype control mAbs were added. In addition, primary mouse keratinocytes (Cellntec) were stimulated with IL-1β for 6 hours and RNA were extracted for analysis of chemokine expression by real-time qPCR. For γδT cell development study, skin, thymus and BM were taken from pups before (E18 or E20) or after birth.
Protocol detail
Skin Cell Isolation and Cytokine Stimulation for Th17 Analysis
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Birth
Buffer
Cell
Cell proliferation
Cfse
Chemokine
Collagenase
Dnase i
Flow cytometry
Hyaluronidase
Il 17
Il 1β
Intracellular
Isotype
Keratinocytes
Mabs
Mouse
Skin
Thymus
Corresponding Organization :
Other organizations : University of Louisville, Ruijin Hospital, Pennsylvania State University
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Variable analysis
independent variables
- RIL-23 (1ng/ml, eBioscience)
- RIL-1β (1ng/ml, eBioscience)
- RIL-23 plus rIL-1β
- Neutralizing mAbs for IL-1β (2μg/ml, eBioscience)
- Neutralizing mAbs for IL-23 (12.5μg/ml, eBioscience)
- Matched isotype control mAbs
- IL-1β stimulation of primary mouse keratinocytes
- Cell proliferation
- Intracellular IL-17
- Chemokine expression in primary mouse keratinocytes
- Whole skin cells prepared from mouse back skin
- Skin cells labeled with CFSE
- Skin, thymus and BM taken from pups before (E18 or E20) or after birth
- Matched isotype control mAbs
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