In Vitro Induction of Pathogenic Th17 Cells into Naive CD4⁺ T-Cells of Mice

To begin, remove the PBS from the pre-coated 24-well plate. Resuspend the enriched mouse splenic naive CD4+T cells in Th17 cell culture medium and dispense into different wells of a 24-well plate pre-coated with anti-CD3. Add Th0 cell culture medium, classical non-pathogenic Th17 culture medium, and classical pathogenic Th17 culture medium for contrast into the remaining wells of the plate.

Add one microliter of anti-CD28 solution to each well. Culture the cells in a 5%carbon dioxide incubator at 37 degrees Celsius for five days. On day two, replace half of the cell culture supernatant medium with fresh culture medium.

Observe the cells under an optical microscope on day five. Collect the cell supernatants from each group and cryopreserve them at 80 degrees Celsius. Naive CD4+T cells were cultured with Th0 medium, and Th17 differentiation medium for five days, resulting in T cells showing cluster growth in both media.

Flow cytometry analysis revealed that 90%of naive CD4+T cells successfully differentiated into pathogenic Th17 cells under the stimulation of a new Th17 cell culture medium.