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Introduction to ELISPOT, with detailed procedure, sample preparation and troubleshooting
ELISPOT is a technique related to ELISA that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is also called enzyme-linked immunospot.
ELISPOT is performed using a PVDF or nitrocellulose membrane 96-well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein.
The resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags. An advantage to using fluorescence is the ability to identify more than one secreted protein at a time.
The membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence, thus this is a quantitative method for evaluating protein secretion.
In the analysis software, set the following parameters for measurement:
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.
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.
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:
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 FicollTM, 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:
Dilute whole blood sample with an equal volume of sterile NaCl 0.9% or balanced salt solution such as PBS or HBSS.
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.
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.
Rapid freezing damages cells.
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.
Check for false positives by running a media negative control