I am planning to do MTS assay so before that, I need to add different concentrations of blocking agent. I used the gelatin coated wells for seeding. After 24 hours of seeding, I washed with PBS with Ca and Mg. Only 10% is left. After 48 hours of seeding, 50% is left with signs of cells not attaching and nearly falling apart. If the seeding time becomes more than 48 hours, I am also afraid the cell conditions will not change that much even though I add different concentrations. FYI: I added 960x1000 cells in one 24-well. After 24 hours, cell confluency is around 80% and seem to be well attached.