I recently performed an experiment where I coated a CD3 antibody on a 96 well (flat-bottom, TC treated) plate at a concentration of 4 µg/ml (50 µl per well) for 2h at 37°C, 5% CO2. Afterwards, I rinse my plate 1x with PBS and plate my cells asap.

This is something that I do routinely, and my specific type of cells always reacts in a very standard/comparable way (strong activation together with CD28 4 µg/ml). However, this time my cells were not activated properly despite coating my plate with CD3 and adding CD28 in the culture media.

The only difference between now and previous times is that after coating my plate with CD3 for 2h, I rinsed my wells and then left my plate with the coating for another 2-3h in the flow without any fluid in the wells. Extra info: the antibody is not expired and all other steps were performed exactly as in previous experiments (no temperature variations, same incubation time ect.)

Could this explain why my cells were not properly activated? And does anybody have experience with storing a CD3-coated plate for a longer period of time?

Thank you in advance for all input.