Mouse th17 cell differentiation kit

Il17a-GFP knock-in mice (C57BL/6-IL17atm1Bcgen/J) were purchased from Jackson Laboratory. These mice express enhanced GFP (eGFP) as a marker of IL17a activity. Splenic naïve CD4+ cells (CD4+ CD44^loCD62L^hi) were isolated using EasySep Mouse Naïve CD4+ T Cell Isolation Kit (StemCell Technologies) from IL-17A-eGFP- mice and cultured in Th17 polarizing condition for 4 days using Mouse Th17 Cell Differentiation Kit (R&D). eGFP+ CD4+ live T cells were FACS sorted by a FACSAria II (BD Biocsiences) and injected (1 × 10⁶ cells per mouse) IV into SFB⁻ JAX WT and Tnf−/− recipient mice previously treated with vehicle or cPTH for 6 days. 3 days later eGFP+ CD4+ T cells in BM of recipient mice were analyzed by flow cytometry.
WT and Tnf −/− spleen T cells were purified by negative immunoselection using MACS Pan T cell isolation kit (Miltenyi Biotech). These cells were injected (5 × 10⁶ cells per mouse) I.V. into TCRβ−/− recipient mice 2 weeks before treatment. Successful T cell engraftment was confirmed by flow cytometry of the spleens of the recipient mice harvested at sacrifice.

Il17a-EGFP–knockin mice (C57BL/6-IL17atm1Bcgen/J) express EGFP as a marker of IL-17A activity. Naive CD4⁺ T cells (CD4⁺CD44^loCD62L^hi cells) were isolated from the spleens of Il17a-EGFP mice using the EasySep Mouse Naive CD4⁺ T Cell Isolation Kit (STEMCELL Technologies). EGFP^– naive CD4⁺ T cells were cultured in Th17-polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP⁺CD4⁺ Th17 cells. EGFP⁺CD4⁺ live Th17 cells were FACS sorted by a FACSAria II (BD Biosciences) and injected i.v. (1 × 10⁶ cells per mouse) into SFB^– JAX WT and Tnf^–/– recipient mice, which had been subjected to fractures 2 days before the T cell transfer. One day after transfer, the relative and absolute frequencies of EGFP⁺CD4⁺ T cells in the callus of recipient mice were determined by flow cytometry.

Naïve CD4⁺CD25^-62L^hiCD44^lo T cells were isolated from pooled single-cell suspensions of spleen and dLNs by a BD FACS Aria III flow cytometer (BD Biosciences). Purified naïve T cells were stimulated with plate-bound anti-CD3 mAb (R&D) for 5 days in the presence of polarizing cytokines and blocking antibodies, according to the manufacturer’s protocol of the mouse Th17 cell differentiation kit (R&D, Cat# CDK017). We cultured 1.25 × 10⁵ cells/well in 96-well plates with a total volume of 0.2 ml/well of culture medium. The mouse Th17 differentiation media were refreshed on day 3 by adding an equal volume of Th17 differentiation fresh media. The IL17A productions in the supernatants were measured by ELISA following the manufacturer’s instructions.