Enzyme-linked Immunospot Technique ( ELISPOT ) Protocol
Enzyme -linked immunospot (ELISPOT) is a technique related to ELISA that is used to detect secreted proteins such as cytokines and growth factors .
ELISPOT uses a 96-well plate with a PVDF or nitrocellulose membrane pre-coated with antibodies specific for the secreted protein. Cells are first added to the plate and attach to the coated membrane. The cells are then stimulated and the secreted protein binds to the antibody. Next, a detection antibody is added that specifically binds to the immobilized protein.
The resulting antibody complex can be detected by enzymatic production of a colored substrate or by using a fluorescent label. The advantage of using fluorescence is that it can identify multiple secreted proteins at once.
The membrane can be analyzed by counting spots manually or using a specially designed automated reader. Each secreting cell appears as a single color or fluorescent spot, so this is a quantitative method for assessing protein secretion.
1. Plate Preparation and Primary Antibody Coating
- PVDF membrane
- 96-well plates
- 35% ethanol
- PBS
-Capture Antibody
- 2% skimmed milk powder
Experimental Steps
1. Place the PVDF membrane into a 96-well plate and incubate in 35% ethanol for 30 seconds.
Note: Wash thoroughly with PBS to ensure complete removal of ethanol. Any residual ethanol will affect cell viability and antibody binding.
2. Coat a 96-well plate with capture antibody diluted in phosphate buffered saline (PBS) .
Tip: To obtain well-defined spots, use approximately 0.5-1 µg of antibody per well.
3. Incubate overnight at 4°C .
4. Empty the wells, pat dry, and wash with PBS.
Warning: Do not use a microplate washer at this stage.
Note: ELISPOT plates are more fragile than ELISA plates and should be handled with care. Be gentle when patting dry.
5. Add 100 µl of 2% skim milk powder to each well to block nonspecific binding to the membrane. Incubate the plate at room temperature for 2 hours.
6. Wash the plate 3 times with PBS and let dry.
Tip: If desired, the plate can be stored at this point. Place the plate in a sealed plastic bag with desiccant and store at 4°C for no longer than 2 weeks.
2. Sample preparation
Most ELISPOT experiments are performed with isolated PBMCs (peripheral blood mononuclear cells). Both freshly prepared cells and cryopreserved cells can be used for the assay. However, it is recommended to allow the frozen cells to sit for at least an hour after thawing to allow for the removal of cellular debris before adding to the plate.
To preserve cell function, PBMCs should be prepared and plated within 8 hours of blood sample collection. If blood samples are left for longer than this, granulocytes (neutrophils) mixed with PBMCs may become activated. This may change their buoyancy curve when PBMCs are separated from granulocytes using FicollTM, so granulocytes may contaminate the PBMC layer. Activated granulocytes may also begin to activate some PBMCs (they can downregulate the signaling zeta chain of CD3, inhibiting T cell function).
If preparation and plating cannot be completed within 8 hours:
Dilute the blood sample immediately. This helps minimize granulocyte contamination. For example, dilute 1:1 in RPMI or PBS. Store at room temperature (not 4°C ).
Granulocytes are removed by cross-linking erythrocytes and granulocytes and then separated from PBMCs using FicollTM. (Disadvantage: some PBMCs may be lost). There are commercially available kits for this purpose.
Ship fresh samples at ambient temperature. Please note that the shipping temperature may be below 4℃. There are commercially available containers that can keep samples at ambient temperature.
Freezing of samples should be optimized. Serum-free media should be used whenever possible (serum contains mitogens and inhibitors that may affect results).
1,Preparation of PBMCs from fresh blood samples
Peripheral blood monocytes (PBMCs) from fresh blood by density gradient separation according to the Ficoll-PaqueTM manufacturer's protocol.
Experimental Steps
1. Dilute the 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 on a volume of Ficoll-Paque equal to the initial blood volume.
NOTE: Be careful to minimize mixing of sample and Ficoll-Paque™.
3. Centrifuge at 2400 rpm for 20 minutes at room temperature with the brake off.
4. Remove PBMCs from the interface between the Ficoll-Paque and plasma layers.
5. Wash the enriched cells with sterile NaCl 0.9% and centrifuge twice for 5 min each at 2000 rpm and then at 3000 rpm for 8 sec to remove platelets.
6. Adjust the recommended cell density in the culture medium to suit the subsequent ELISPOT procedure.
7. Freeze cells for storage (Optional).
( 1 ) Prepare 20% DMSO in cell culture medium. Keep on ice.
( 2 ) Label the cryovials.
( 3 ) Resuspend the cells in ice-cold culture medium at a density of 40×106 cells/mL. Place on ice.
( 4 ) Aliquot 0.5 mL of the cell suspension into cryovials.
( 5 ) Gently add 20% DMSO to the cell suspension at a ratio of 1:1. The final suspension concentration is 20 × 106 cells/mL.
( 6 ) Immediately place the cryovials into a freezing container.
Warning: Rapid freezing can damage cells.
Tip: To check for normalization of results, keep a large frozen sample of cells from a good quality donor to use as a control in a series of ELISPOT experiments.
2,Thawing frozen cells for ELISPOT assay
Experimental Steps
1. Thaw cells quickly in a 37 °C water bath or beaker of warm water.
Warning: Do not vortex cells at any time.
2. Gently transfer the cells to a 50 mL tube containing 15 mL of culture medium. Use 0.5 to 5.0 mL of cells per 50 mL tube.
3. Fill the tube with medium to a volume of 50 mL. Gently invert the tube to mix.
4. Spin cells at 1200 rpm for 5 minutes.
5. Pour off the supernatant and gently flick the tube to resuspend the pellet. Count and adjust the cells to the desired density in appropriate culture medium.
6. The cells are now ready for ELISPOT assay.
3,Preparation of mouse spleen samples
Experimental Steps
1. Tissue homogenization.
1.1 Gently tease the tissue using a sterile stainless steel tube and disperse it into 30 mL of the recommended culture medium.
1.2 Additionally, disperse clumps by gently pipetting up and down several times.
1.3 Remove remaining cell clumps and debris.
2. Centrifuge the cells at 2000 rpm for 5 minutes and resuspend the cell pellet in the recommended medium for the following steps.
Tip: Consistent results can be obtained if splenocytes are pre-stimulated for 24-48 hours with appropriate stimuli to allow cytokine release before adding them to the ELISPOT plate and further incubated for 8-16 hours with the same supplements to allow spot formation.
3. Cell Incubation
Experimental Steps
1. Use the PBMCs prepared in the previous stage. Count the cells using a viability dye such as trypan blue. The viability of cells should be more than 95%.
2. Dilute the cells to the desired concentration and add the cell suspension to the wells.
Tips: If you want to optimize cell number, use a 1:2 dilution series. Do not shake the dish.
The number of cells per well should be optimized. For example, if a low percentage of cells are expected to secrete the target cytokine, use more cells. Refer to the specific target kit protocol for recommendations on assay controls and number of cells per well. Typically, the number of cells should be between 2 × 105 and 4 × 105 PBMC cells per well.
If possible, use serum-free media, as serum contains many proteins that may affect the results. Alternatively, several batches of serum can be tested to find the one with the best response-to-noise ratio. This batch can then be stored and used in subsequent experiments.
3. Incubate overnight in a 37°C CO2 incubator. Do not shake the dish.
Tips: During the overnight incubation, cells will secrete cytokines, which will bind to the primary antibody.
If cells need time to respond to stimulation, see the indirect method below.
Warning: If you have multiple cell plates , do not stack them to prevent edge effects.
Note: Do not move the culture dish while the cells are incubating. This will result in unclear "snail trail" spots.
4. Indirect ELISPOT (Optional)
If cells take some time to respond to the stimulus, it may be necessary to pre-treat them with the stimulus in a separate 96-well dish before transferring to the ELISPOT plate.
Positive control stimulation
Experiments using ELISPOT to detect cytokines require the use of positive controls. In these positive control wells, cells should be stimulated with an agent known to induce expression of the cytokine being tested. This can then be compared to negative controls (either untreated or stimulated with a different secreted protein).
Typical stimuli include:
- LPS stimulates the secretion of IL - 1β and IL - 6
- PMA and ionomycin stimulate the secretion of IL - 2 and IL - 4
- PHA, 10 µg/mL, for interferon gamma
- Anti-CD3/CD28 antibodies against IFNγ, IL - 4, IL - 10 and Granzyme B
Tip: Make sure you stimulate PBMCs with the correct stimulator to detect your cytokine of interest.
5. Incubate with PBS 0.1% Tween 20 for 10 minutes to wash away cells and unbound cytokines.
Warning: Do not use a microplate washer at this stage.
Note: Make sure to include Tween 20 in the wash buffer. Some cells will begin to attach after overnight culture (e.g. some stem cells). Tween 20 will help wash these cells off the membrane.
4. Incubate with detection antibodies
Materials required
- Conjugated detection antibody
- PBS 0.1% Tween 20
Experimental Steps
1. Dilute the conjugated detection antibody in PBS 1% BSA.
2. Add the conjugated detection antibody to the wells and incubate at room temperature for 1 - 2 hours.
Note: Incubation time may need to be optimized.
3. Wash the plate three times with PBS 0.1% Tween 20 to remove non-specific detection antibody binding.
5. Enzymatic Detection
Experimental Steps
1. Add enzyme substrate solution to each well.
For enzyme detection protocols, the base should be removed from the bottom of the plate to allow for thorough washing of the membrane prior to addition of substrate/chromogen.
For example, after incubation with streptavidin-alkaline phosphatase conjugate, remove the base and wash both sides of the membrane under running distilled water. This helps prevent high background because some reagents may leak through the membrane into the bottom tray of the plate.
2. After replacing the base and adding substrate, carefully monitor the formation of spots.
3. Once the reaction slows down, gently wash the plate with PBS 0.1% Tween 20 to stop the reaction.
4. Take the base off the plates and wash both sides of the membrane with distilled water to prevent spot formation.
5. Wipe the culture plate dry and allow the membrane to dry at room temperature. It can then be tested and analyzed.
Note: If the membrane is stored overnight at 4℃, the spots may become clearer and may continue to improve for up to 2 weeks. If storing, wrap the membrane in foil and keep it at 4℃.
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