- High background
- No spots/very few spots
- Blank areas
- Blank center
- False positives
- Confluent spots
- Poorly defined spots
Perform wash steps carefully.
Wash both sides of the membrane with distilled water before and after color development. Some reagents may leak through the membrane into the base of the plate, and these can cause high background if not washed away.
Too many cells secreting cytokine/protein of interest
Reduce the number of cells per well. This will require optimization. You may also need to optimize the concentration of stimulant used.
Plate not dried properly
Dry the plate longer before reading. Drying overnight at 4°C may help increase the contrast between background and spots.
Over developed plate
Reduce developing time.
Not enough cells secreting cytokine/protein of interest
Increase the number of cells. This will require optimization.
Ensure the cells are stimulated correctly
Also use a positive stimulation control – a stimulant that you know will induce expression of your cytokine/protein of interest.
Cells not incubated for long enough or may take time to respond to stimulant
Increase the cell incubation time or use indirect method (pre-treat cells with stimulant)
Inadequate color development
Monitor color development with an overhead microscope and ensure that the developing reagents have been stored correctly and have not gone off.
Not enough primary or secondary antibody
Concentration of the primary and/or secondary antibody will need to be increased. This will require optimization.
Membrane not pre-treated
Ensure membrane is adequately pretreated with 70% ethanol. Wash well with PBS 3X afterwards.
Membrane has dried out at some stage
Ensure membrane does not dry.
Cells unevenly distributed
Ensure you mix the cells gently to have a good homologous cell suspension before pipetting out into the wells.
Damage from washing
Flow rate on automated washer (or pipetting) may be too high. Need a more gentle washing procedure
Secondary antibody aggregates
Filter the secondary antibody
Cells still on the membrane
Ensure all the cells are washed from the membrane with PBS Tween 20 before secondary antibody incubation. Cells left on the membrane will give irregular shaped spots.
Cell culture contamination
Keep reagents as sterile and clean as possible. Ensure your cell culture technique is aseptic. Check for false positives by running a media negative control. For plate movement, see poorly defined spots
Poor coating, too much antibody
Reduce primary antibody concentration
Prolonged cell culture
The longer the cells are incubated, the more cytokine/protein they will secrete. This will result in larger spots that will start to merge and become indistinguishable. Reduce cell culture step incubation time.
Over-stimulation will result in a lot of cytokine/protein being secreted by the cell. This will produce spots that will start to merge and become indistinguishable. Reduce the amount of stimulant in the culture media or culture for a shorter amount of time.
Membrane not pre-treated.
The membrane must be pre-treated with ethanol or this can result in fuzzy, poorly defined spots. It will be difficult for the reader to distinguish these.
Plate movement during cell incubation
Do not allow the plate to move during cell incubation as cells that have moved will create more than one spot. If possible use a dedicated incubator that will not be opened during the incubation. Do not tap the plate after adding cells.