I sometimes get streaks and irregular spots in the well bottoms. What could be causing this?

T cells can actively migrate during the assay’s duration. In particular, T cells that have been preactivated in vivo or in vitro can migrate rapidly, leaving a cytokine trail. Also, if the plates are shaken (due to banging of the incubator door, for example) or moved during the capture period, the cells will roll, leaving streaks and irregularly shaped spots.

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