T Cell Activation with anti-CD3 Antibodies Protocol - Mouse


Reagent List

  • Sterile PBS
  • Anti-mouse CD3ε, clone 145-2C11 (Ultra-LEAF™ format, Cat. No. 100339)
  • Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS and 2mM L-glutamine)
  • Sterile, single-cell suspension (e.g., splenocytes, lymph node 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 5µg/ml solution of anti-CD3ε (clone 145-2C11) 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 50µl of sterile PBS.  

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

  4. Aseptically decant antibody solution from microwell plate.  

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

  6. Prepare single cell suspension of cells of interest.  

  7. Resuspend cells in supplemented cell culture medium to 1-2 x 106/ml.  

  8. 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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