實驗概要
Th9 cells are a subpopulation of T-helper cells, characterized to be involved in allergic diseases and resistance against intestinal nematodes. Unlike Treg cells, Th9 cells are not suppressive, as they can promote T cell proliferation along with other effector T cells. Th9 cells do not express any well-defined transcription factors like T-bet, GATA3, RORγt and Foxp3, clearly differentiating them from Th1, Th2, Th17 and Foxp3 iTreg populations. TGF-β reprograms Th2 T-helper cells to lose their characteristic profile of IL-17 secretion and switch to IL-9 secretion. Differentiation of Th9 cells can be obtained by a combination of TGF-β and IL-4. In addition, the neutralization of IFN-γ is critical to drive the pathway to Th9 cells. Here we provide a effective protocol for the generation of mouse Th9 cells in vitro.
主要試劑
Sterile PBS
Cell culture medium
Sterile plastic petri dishes
RBC Lysis Buffer [Details: Biolegend, cat. # 420301]
Anti-mouse CD3ε, clone 145-2C11 [Details: Biolegend, LEAF? format, cat. # 100314]
Anti-mouse IFN-γ [Details: Biolegend, LEAF? format, cat. # 505812]
Recombinant mouse IL-4 [Details: Biolegend, cat. # 563202]
Recombinant human TGF-β1 [Details: Biolegend, carrier-free] [cat. # 580702]
Recombinant mouse IL-2 [Details: Biolegend, cat. # 575402]
Brefeldin A [Details: Biolegend, cat. # 420601]
PMA [Details: Sigma cat. # P8139]
Ionomycin [Details: Sigma cat. # I0634]
實驗步驟
1. Coat 60 × 15 mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5μg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
2. Plate splenocytes at 1 x 106/ml. Culture cells for 3 days in presence of anti-mouse CD28, clone 37.51 (5 μg/mL), recombinant TGF-β1 (10 ng/mL), recombinant mouse IL-4 (10 ng/mL), recombinant mouse IL-2 (20 ng/ml), and anti-mouse IFN-γ (10 μg/ mL).
3. On day 3, wash cells once and then restimulate in complete medium with 500ng/ml PMA and 500 ng/mL ionomycin, in the presence of Brefeldin A for 6 hours.
4. After harvesting, the cells are ready for staining.