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, keeping it protected from light. Drying overnight at 4oC may help increase the contrast between background and spots.
Reduce developing time.
Exceeding 1 hour incubation with the enzyme substrate may result in increased background color.
No spots/very few spots
Not enough cells secreting cytokine/protein of interest
Increase the number of cells. Cell numbers should usually range between 1- 2 X 105 cells per well, and this may require some 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 lost activity.
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 35% ethanol. Wash well with PBS 3X afterwards.
Membrane not washed adequately after ethanol treatment
Wash the membrane thoroughly. Sometimes this can occur if ethanol is trapped between the membrane and the bottom of the plate (leakage).
Membrane has dried out at some stage
Ensure membrane does not dry.
Cells unevenly distributed
Mix cells gently to obtain a homogenous suspension before pipetting the cells into the wells.
Pipette tip touched the membrane
Take care with pipetting steps, particularly with washing, automated washing in particular.
Formation of foam
During washing, foam formation can occur. Squirt bottles with narrow spouts produce excessive foam, preventing an effective and uniform wash.
Damage from washing
Flow rate on an automated washer may be too high, or manual pipetting may be too harsh. Requires a gentler washing procedure.
Check for false positives by running a media negative control
Secondary antibody aggregates
Filter the secondary antibody.
Cells still on the membrane, cell debris
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.
Contaminating platelets (when using PBMC prepared from blood samples)
PBMC preparation needs to be efficient. Wash the plate well after cell culture stage.
Cell culture contamination (dust and microbial)
eep reagents as sterile and clean as possible. Ensure your cell culture technique is aseptic. Reagents can be filtered using a low protein binding syringe 0.2 µm pore size.
Mitogens and other factors in the serum are stimulating the cells
Heat inactivate the serum
Also see “Poorly defined spots” regarding plate movement
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. (It is generally advised not to exceed 24 hours).
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.
Poorly defined spots
Membrane not pre-treated
The membrane must be pre-treated with ethanol or the result may be 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.
Coating antibody not concentrated enough
Increase coating antibody concentration.
Spot quality can help to define if the capture antibody is too diluted or if there is an issue during the coating step.
White spots in the middle of a normal spot (more usual with enzymatic detection)
This means the enzymatic conjugate has run out of substrate. Therefore a higher concentration of second antibody and substrate is required.
For fluorescence – increase the antibody concentrations.
Inconsistency in results between wells
Do not stack the plates during cells incubation. This will give an edging effect within the plate due to non-uniformity of temperature distribution.
Ensure a well mixed single cell suspension of sample is used when adding cells to the wells.