Pre-migration/invasion reference wells are used to establish the size and position of the detection zone in order to quantify the extent of movement in experimental wells. For Oris assays, you can leave some stoppers in place until you read the results. Since Oris Pro assays lack stoppers, this method is not applicable. For Oris Pro and Oris assays, alternative methods to establish reference wells are: Simply collect images at time zero as migration is about to begin. Add fixative to replicate wells or a replicate plate at the beginning of the assay. However, when fixing cells in a subset of wells in the plate, this method risks impacting cells adjacent to fixed wells as they may be exposed to fixative vapors. Add an inhibitor of migration or invasion, such as cytochalasin D, to reference wells at the beginning of the assay. Be sure to add sufficient inhibitor to block cell movement completely. You may wish to read a related application note here.