I am performing CBA for the first time. I have stimulated my cells (HTP1-derived macrophage) in two ways:

1. Cells were stimulated by incubating with samples for 3 hours then LPS was added.

2. Cells were incubated with LPS for the same 3 hours and then samples were added.

The cells were stimulated with the positive control which in this case is LPS+medium was added to the control wells after 3 hours of incubation which is relative to the first condition. My question is positve control will work for the first condition due to same time of stimulation but will it also work for the second condition? If not what is your suggestion?

Thanks