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Blank areas in ELISPOT

Find solutions for blank areas in ELISPOT.

Cells unevenly distributed

Possible causes

Solution

Cells have been distributed unevenly throughout the sample.

  • Mix cells gently to obtain a homogenous suspension before pipetting the cells into the wells.

Formation of foam

Possible causes

Solution

During washing, foam formation can occur.

Squirt bottles with narrow spouts produce excessive foam, preventing an effective and uniform wash.

  • During washing, foam formation can occur. Squirt bottles with narrow spouts produce excessive foam, preventing an effective and uniform wash.

Membrane has dried out at some stage

Possible causes

Solution

Membranes can dry out at many experiment stages if not carefully managed.

  • Ensure membrane does not dry.

Membrane not pre-treated

Possible causes

Solution

You may not have pretreated your membrane in the course of your experimental preparation.

  • Ensure membrane is adequately pretreated with 35% ethanol. Wash well with PBS 3X afterwards.

Membrane not washed adequately after ethanol treatment

Possible causes

Solution

Sometimes this can occur if ethanol is trapped between the membrane and the bottom of the plate (leakage).

  • Wash the membrane thoroughly. 

Pipette tip touched the membrane

Possible causes

Solution

Pipettes can touch the membrane at many points during your experiment.

  • Take care with pipetting steps, particularly with washing, automated washing in particular.