T Cell Activation with anti-CD3 Antibodies Protocol - Human

 

Reagent List

Sterile PBS Anti-human CD3 Antibody: Clone UCHT1 (Ultra-LEAF™ format, Cat. No. 300437) Anti-human CD3 Antibody: Clone OKT3 (Ultra-LEAF™ format, Cat. No. 317325) Note: Soluble forms of Ultra-LEAF™ purified UCHT1 (1µg/ml) or Ultra-LEAF™ purified HIT3a (0.01 - 0.1µg/ml) may be used to activate T cells from PBMC cell populations.

Anti-human CD3 Antibody: Clone HIT3a (Ultra-LEAF™ format, Cat. No. 300331) 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 (e.g., Costar® Cat. No. 3596)

 

Protocol Steps

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1. Prepare a 10µg/ml solution of anti-CD3 (clone UCHT1, OKT3, or HIT3a) in sterile PBS.  


2. Dispense 50µl 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 200µl cell suspension into plate microwells. Cover with lid. Incubate at 37°C in 5% CO² and 100% humidity for 3 days.  

General Tips & FAQ

• What if I want to stimulate my cells to a particular T-cell lineage?
  • As a guide for this, you can take a look at our Activation Bundles page, which will provide you with the listing of our products you will need to differentiate into different T-helper cell lineages.