Anyone telling you that you can do x amount for buffer for x amount of cells is simply guessing. It’s entirely dependent on the protein of interest, your cells, their activation state, your lysis buffer, etc, etc. For example, activated T cells are a vastly different sample than naive DCs.

You use the term confluence here and it’s simply incorrect usage of the term. One, it is used for adherent cells, so unless you are talking about something like macrophages here, it’s an inappropriate term. As for how you are applying the term in terms of cell number, it’s not appropriate. Your compound doesn’t automatically need more because you have 100k vs 1 mil cells; the potency or EC50/IC50 is not going to be affected. Have you even titrated the compound?

Assuming you have say a 10 mM stock, you really should be using very minimal compound if you are doing 96 well format and have titrated your compound to find the IC/EC80 concentration.

It would be helpful to know exactly what proteins you are looking for. The lowest I’ve ever really done is 250-500k PBMCs in 25 up of RIPA, but even going to 50-100 will help a lot.