Which wells should be considered “blank” or “negative” while creating your layout?

ELISAAnalysis uses “Negatives” for calculating the Background Cut-off Threshold.  So if you feel that your control could be a factor for potential noise in the experiment, then you should specify your controls as “Negatives” in the plate layout. Please note that “Blanks” are completely ignored for calculations in our software.  A knowledge base article on background cut-off could be helpful for you before you proceed to calculation step and can be found here: http://elisaanalysis.com/knowledge-base/elisa-software-background-cut-off-or-threshold-calculation/