实验概要

T-cell  activation, which plays a central role in the regulation of immune  responses, involves multiple intracellular signaling events originating  from the cell surface TCR/CD3 complex. Cross-linking of the TCR/CD3  complex by immobilized anti-CD3 antibody induces T-cell activation,  leading to the production of cytokines such as interleukin 2 (IL-2).  IL-2 binds to its high-affinity receptor to promote cell proliferation.  Additionally, co-stimulatory surface molecules such as CD28 have been  shown to provide accessory signals in T-cell activation, enhancing IL-2  production when co-stimulatory anti-CD28 antibody is combined with  plate-bound anti-CD3

主要试剂

Sterile PBS

Anti-human CD3, clone UCHT1 (LEAF™ format, Cat. No. 300413/4) or clone HIT3a (LEAF™ format, Cat. No. 300313/4)

Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS and 2mM L-glutamine)

Sterile  single-cell suspension of Ficoll-Hypaque-purified peripheral blood  mononuclear cells, isolated T cells, or T cell subsets

96-well flat-bottom tissue culture plates with lids 

实验步骤

1. Prepare a 10 µg/ml solution of anti-CD3 (clone UCHT1 or HIT3a) in sterile PBS.

2.  Dispense 50 µl of the antibody solution to each microwell of the  96-well assay plate. For the unstimulated control wells, add 50 µl of  sterile PBS.

3. Seal plate. Incubate at 37°C for 2 hours or 4°C overnight.

4. Aseptically decant antibody solution from the microwell plate.

5. Wash plate microwells 3 times with sterile PBS. Discard liquid.

6. Prepare single cell suspension of cells of interest in supplemented cell culture medium to 1-2 x 10⁶/ml.

7.  Aliquot 200 µl cell suspension into plate microwells. Cover with lid.  Incubate at 37°C in 5% CO2 and 100% humidity for 3 days.

*  Soluble forms of LEAF™ purified UCHT1 (1 µg/ml) or LEAF™ purified HIT3a  (0.01 – 0.1 µg/ml) may be used to activate T cells from PBMC cell  populations.