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Inconsistent signals across plates are most commonly caused by temperature differences between the plate and reagent at the time of addition (ensure both are equilibrated to room temperature for 30 minutes before mixing), insufficient cell lysis (increase mixing time or speed), or signal read outside the stable window (read plates within 10 minutes to 1 hour after stabilization)

Bhansali S, Bhansali A, Dutta P, Walia R, Dhawan V