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

  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.

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