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Th9 Polarization of Mouse Splenocytes

2019.4.22

实验概要

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.

主要试剂

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]

实验步骤

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.


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