Physical Membrane Damage and Artifacts (ELISPOT)

Question

Accepted Answer

Membrane shows physical damage, scratches, or irregular patterns. Spots may appear distorted or artifacts present as non-specific signals. Common causes: 1) Flow rate on automated washer too high or manual pipetting too harsh 2) Pipette tips touching the membrane during washing or reagent addition steps 3) Membrane dried out at various experimental stages 4) Membrane not pretreated with 35% ethanol before coating Solutions: 1) Use gentler washing procedure; reduce flow rate on automated washers 2) Exercise care during all pipetting steps, especially automated washing 3) Keep membrane hydrated at all stages; never allow to dry out 4) Pretreat membrane with 35% ethanol followed by 3× PBS washes