ELISPOT sample preparation tips
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 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 4oC).
- Remove the granulocytes by cross-linking red blood cells and granulocytes, then separate from PBMC using FicollTM. (Disadvantage: some PBMC may be lost). There are commercially available kits for this.
- Ship fresh samples at ambient temperature. Note that transport temperatures can be below 4oC. Containers are commercially available which keep samples at ambient temperature.
- 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-PaqueTM manufacturer's protocol)
- Dilute whole blood sample with an equal volume of sterile NaCl 0.9% or balanced salt solution such as PBS or HBSS.
- Layer the diluted sample over a volume of Ficoll-Paque RTM equivalent to the initial blood volume. Be careful to minimize mixing of the sample and the Ficoll-PaqueTM.
- Centrifuge for 20 minutes at 2400 RPM at room temperature with the brake off.
- Remove the PBMC from the interface between the Ficoll-PaqueTM and the plasma layer.
- 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.
- Adjust cell density in the medium recommended for the following ELISPOT procedure.
Preparing mouse spleen samples
- 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.
- 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.
- Make up 20% DMSO in cell culture media. Keep on ice.
- Label cryovials.
- Re-suspend cells at 40 x 106 cells/mL in ice-cold medium. Keep on ice.
- 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.
- 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
- Thaw cells quickly in a 37oC water bath or beaker of warm water. Do not vortex cells at any time.
- 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
- Fill tube to 50 mL with culture media. Gently invert tube to mix.
- Spin down cells at 1200 RPM for 5 minutes.
- Pour off supernatant and flick tube gently to re-suspend the pellet. Count and adjust cells to desired density in appropriate medium.
- Cells are now ready to use in the ELISPOT assay.