Detection of Th17 (3-color) in Mouse Spleen

Tips:

1. After the preparation and counting of the splenic single-cell suspension are completed, it is necessary to perform stimulation and inhibition culture using cytokine stimulation inhibitors (for experimental conditions of stimulation and inhibition, please refer to the instructions of the reagent kit used).

2. PMA stimulation can induce partial internalization of CD4 on the surface of human T cells. Therefore, it is necessary to select the CD4 clone RM4-5, which has minimal impact on internalization.

3. Isotype controls are required for IL-17A because cytokine expression levels are generally low.

4. Th17 cells are characterized by the CD3⁺CD4⁺IL-17A⁺ phenotype.

5. Permeabilization reagents can cause significant damage to cells. It is recommended to first resuspend the cell pellet into a single-cell suspension after centrifugation before adding the permeabilization reagent, in order to minimize cell loss.