ELISPOT introduction and protocol

General ELISPOT procedure

1. Prepare PVDF membranes in the 96-well plates by incubating in 35% ethanol for 30 s.
   
   

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   Ensure the ethanol is completely removed by washing thoroughly with PBS. Any remaining ethanol can affect cell viability, as well as antibody binding. 
   
   

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2. Coat 96-well plate with capture antibody diluted in phosphate buffered saline (PBS). Incubate overnight at 4°C.
   
   

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   Approximately 0.5–1 µg per well of antibody should be used for well-defined spots. Kits are optimized with capture concentrations for best performance (100 µl per well).
   
   

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3. Empty the wells, tapping them dry, and wash with PBS. 
   
   

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   ELISPOT plates are more delicate than ELISA plates and should be handled with care. When tapping dry, do so gently. Do NOT use a plate washer at this stage.

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4. Add 100 µl per well 2% dry skim milk to block non-specific binding to the membrane. Incubate the plate for 2 h at room temperature.
5. Wash the plate 3 times in PBS and leave to dry. If necessary, the plates can be stored at this stage. Store at 4°C for not more than 2 weeks, in a sealed plastic pouch with desiccant.
6. Prepare peripheral blood mononuclear cells (PBMCs) from fresh blood using a Ficoll™ gradient. Count the cells using a viability dye like trypan blue. They should be over 95% viable. Dilute cells to the required concentration and add the cell suspension to wells. If optimizing the assay for cell number, use a 1:2 dilution series. Do not shake plates.
   
   The number of cells per well should be optimized. For example, use more cells if a low percentage of cells are expected to secrete the target cytokine. Refer to the specific target kit protocols for recommendation on assay controls and cell number per well. Typically cell numbers should usually range from between 2x10^​5 to 4x10⁵ PBMC cells per well. 
   
   If possible, use serum-free media as serum contains many proteins which could affect the results. Alternatively, several batches of serum can be tested to find one with optimal response to noise ratio. This batch can then be stored and used in subsequent experiments.
7. Culture overnight at 37°C in CO_² incubator. Do not shake the plates.
   
   

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   Do not move the plates while the cells are culturing. This will lead to 'snail trail' spots that will not be well defined. Don't stack the plates if you have more than one to prevent edge effects
   
   

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8. During the overnight incubation the cells will secrete cytokine, which will bind to the primary antibody.
   
   

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   If cells take time to respond to stimulation, please see indirect method below.
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9. Wash away the cells and the unbound cytokine by incubating with PBS 0.1% Tween 20 for 10 min.
   
   

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   Ensure you include Tween 20 in the wash buffer. Some cells will have started attaching after culture overnight (e.g. some stem cells are known to do this). Tween 20 will help wash these off the membrane. Do not use a plate washer at this stage.
   
   

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10. Dilute the conjugated detection antibody in PBS 1% BSA. Add to wells and incubate for 1–2 h at room temperature (the incubation time may require optimization). Wash plate 3 times with PBS 0.1% Tween 20 to remove non-specific detection antibody binding.
    
    

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    For our ELISPOT kits, detection antibody concentrations have been optimized for best results.
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11. Add the conjugated detection antibody. Dilute the antibody in PBS 1% BSA. Incubate for 1–2 h at room temperature (the incubation time may require optimization). Wash plate 3 times with PBS 0.1% Tween 20 to remove non-specific detection antibody binding.
12. Add the enzyme substrate solution to each well.
    
    

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    For enzymatic detection protocols, the base should be taken off the bottom of the plate to enable thorough washing of the membrane before adding substrate/chromogen. For example, after incubation with the streptavidin alkaline phosphatase conjugate, remove the base and wash both sides of the membrane under running distilled water. This helps to prevent high background as some reagents can leak through the membrane into the bottom tray of the plate. 
    
    

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13. After replacing the base and adding the substrate, carefully monitor spot formation. Stop the reaction by gently washing the plate with PBS 0.1% Tween 20 once development appears to slow. Take the base off the plates and wash both sides of the membrane with distilled water to stop the spot formation.
14. Dry the plates and allow the membranes to dry at room temperature.
    
    

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    Spots may become sharper if membranes are stored overnight at 4°C, and may continue to improve for up to 2 weeks. If storing, wrap membranes can be wrapped in foil, keep at 4°C.
    
    

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15. Punch the membranes out of the wells onto a sticky plastic sheet. This step will depend on your reader's requirements. Consult the reader manual. 
16. Measure the sheet and analyze the membrane circles.

In the analysis software, set the following parameters for measurement:

• Size/spot diameter
• Intensity/saturation
• Circularity/shape
• Spot development/slope

These parameters can be saved and used for subsequent experiments for standardized results.

We recommend reading each plate 3 times and averaging the results in order to minimize error in the measurements.

Indirect ELISPOT

If the cells take some time to respond to stimulation, they may require pretreatment with the stimulant in a separate 96-well culture dish before transferring to the ELISPOT plate.

Positive control stimulation

Experiments to detect cytokines using ELISPOT will require use of positive control. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being detected. This can then be used to compare to the negative control (no treatment or stimulation of a different secreted protein).

Ensure you are stimulating the PBMCs with the correct stimulant for detection of your target cytokine.

Typical stimulates include:

• LPS to stimulate IL1β, IL6 secretion
• PMA and ionomycin stimulate IL2, IL4 secretion
• PHA, 10 µg/ml for IFN gamma
• Anti-CD3/CD28 antibodies for IFN gamma, IL4, IL10 and Granzyme B

EPISPOT sample preparation

Most ELISPOT experiments are done with isolated PBMC (peripheral blood mononuclear cells). Both freshly prepared and cryopreserved cells may be used in the assay. However, it is recommended to let frozen cells rest at least one hour after thawing to allow removal of cell debris before addition to the plate.

PBMCs should be prepared and plated within 8 hours of collecting the blood samples to preserve cell functionality. If the blood samples are left longer than this, the granulocytes (neutrophils) that are mixed with the PBMC can become activated. This can change their buoyancy profile when the PBMCs are separated from granulocytes using Ficoll^TM, and so the granulocytes may contaminate the PBMC layer. The activated granulocytes may also begin to activate some of the PBMCs (they can down-regulate the signal-transducing zeta chain of CD3 which suppresses T cell function.

If preparation and plating are not possible within 8 hours:

1. Dilute the blood sample immediately. This helps to minimize the granulocyte contamination. For example, dilute 1:1 in RPMI or PBS. Keep at room temperature (not 4^oC).
2. Remove the granulocytes by cross-linking red blood cells and granulocytes, then separate from PBMC using Ficoll^TM. (Disadvantage: some PBMC may be lost). There are commercially available kits for this.
3. Ship fresh samples at ambient temperature. Note that transport temperatures can be below 4^oC. Containers are commercially available which keep samples at ambient temperature.
4. Freezing of samples should be optimized. Use serum-free media where possible (serum contains mitogens and inhibitors which could affect the results).

Density gradient separation (according to the Ficoll-Paque^TM manufacturer's protocol)

1. Dilute whole blood sample with an equal volume of sterile NaCl 0.9% or balanced salt solution such as PBS or HBSS.

2. Layer the diluted sample over a volume of Ficoll-Paque R^TM equivalent to the initial blood volume. Be careful to minimize mixing of the sample and the Ficoll-Paque^TM.
3. Centrifuge for 20 minutes at 2400 RPM at room temperature with the brake off.
4. Remove the PBMC from the interface between the Ficoll-Paque^TM and the plasma layer.
5. Wash enriched cells with sterile NaCl 0.9% with 2 centrifugations at 5 minutes each, 2000 RPM, followed by one at 8 seconds at 3000 RPM to remove platelets.
6. Adjust cell density in the medium recommended for the following ELISPOT procedure.

Preparing mouse spleen samples

1. Tissue homogenization: Gently tease the tissue through a sterile stainless steel and disperse into 30 mL of recommended medium. Further disperse clumps by gently pipetting up and down several times. Remove remaining clumps of cells and debris.

2. Centrifuge the cells for 5 minutes at 2000 RPM, and re-suspend cell pellet in medium recommended for the following procedure.

Note: Consistent results can be obtained if the splenocytes are pre-stimulated for 24-48 hours with an appropriate stimuli for cytokine release before addition to the ELISPOT plate with same supplement for a further incubation of 8-16 hours to allow spot formation.

Freezing cells for storage

Rapid freezing damages cells.

1. Make up 20% DMSO in cell culture media. Keep on ice.
2. Label cryovials.
3. Re-suspend cells at 40 x 10⁶ cells/mL in ice-cold medium. Keep on ice.
4. Dispense 0.5 mL cell suspension into cryovials. Add gently into cell suspension 20% DMSO at a ratio of 1:1. Final suspension will be at 20 x 10⁶ cells/ml.
5. Place cryovials immediately into freezing container.

Note: Keeping a large sample of cells frozen down from a good donor to use as a control in a series of ELISPOT experiments can be a useful tool to check standardization of results.

Thawing frozen cells for the ELISPOT assay

1. Thaw cells quickly in a 37^oC water bath or beaker of warm water. Do not vortex cells at any time.
2. Gently transfer cells into a 50 mL tube (0.5 to 5.0 mL of cells per 50-mL tube) containing 15 mL culture media
3. Fill tube to 50 mL with culture media. Gently invert tube to mix.
4. Spin down cells at 1200 RPM for 5 minutes.
5. Pour off supernatant and flick tube gently to re-suspend the pellet. Count and adjust cells to desired density in appropriate medium.
6. Cells are now ready to use in the ELISPOT assay. 

ELISPOT troubleshooting

High background

Wash steps not sufficient

• 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.

​Over-developed plate

• 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

• ​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.

Blank areas

​​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.

Blank centre

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.

False positives

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)

• Keep 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

Confluent 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. (It is generally advised not to exceed 24 hours).

Cells over-stimulated

• 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 cell incubation. This will give an edging effect within the plate due to the non-uniformity of temperature distribution.
• ​Ensure a well-mixed single-cell suspension of sample is used when adding cells to the wells.