Single Cells' Cytokine Secretion Measurement by ELISPOT and ELISA Protocol

Overview

Introduction of ELISPOT and ELISA to Single Cells' Cytokine

Although the enzyme-linked immunospot (ELISPOT) assay is thought to be more sensitive than ELISA, allowing for the detection of a single cytokine-secreting cell out of a million, the major limitation of the ELISPOT assay is that, unlike ELISA, it does not quantitate the amount of secreted cytokines. The output of an ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, and it appears that each individual spot represents a "footprint" of a single cytokine releasing cell.

ELISPOT allows detection of just a few cytokine-releasing cells out of tens of thousands, making this technique a method of choice for vaccine development, cancer research, AIDS research, allergy research, and autoimmune disease studies.

Figure 1. Detection of cytokines (eg: IL-2) released by cells using ELISPOT technique. (Naresh Sachdeva, et al.; 2007)

We worked out a simple protocol for the determination of the average amount of IFN gamma secreted by a single cell. Our protocol can be easily adapted for measuring other cytokines in different species and utilized for both basic research and for developing diagnostic applications.

Materials of ELISPOT and ELISA to Single Cells' Cytokine

Isolation and Culture of Human PBMCs

Ficoll-Paque PLUS.
50 mM Phosphate-buffered saline (PBS), pH 7.2.
Red blood cells lysing solution: 155 mM NH₄Cl, 10 mM NaHCO₃, and 0.1 mM EDTA.
RPMI complete culture medium: RPMI1640 (1 L) supplemented with 50 mL of heat-inactivated fetal calf serum, 1.19 g HEPES, 2 g of sodium bicarbonate, 3.5 μL of beta-mercaptoethanol, and 50 mg of Gentamicin Reagent Solution (see Notes 1 and 2).
Centrifuge capable of spinning 50-mL culture tubes at 500 × g.
Hemacytometer to count lymphocytes under the microscope.
Trypan blue dye.
Microscope equipped with bright-field illumination and phase-contrast optics.
Uncoated, 96-well ELISPOT plates.

ELISPOT Assay

Commercially available ready-to-use ELISPOT assay kit to study secretion of human IFN-γ. Each kit includes a 96-well, PVDF membrane-backed plate precoated with capture antibody, a concentrated solution of detection antibody, a concentrated solution of streptavidin conjugated alkaline phosphatase, BCIP/NBT chromogenic substrate, and wash and dilution buffers.
Blocking buffer to block membranes in plates uncoated with anti-IFN gamma antibodies: PBS with 1% BSA and 5% sucrose.
Mitogens to stimulate release of cytokines from cultured peripheral blood mononuclear cells (PBMCs): Calcium ionomycin (CaI) and phorbol 12-myristate 13-acetate (PMA).
Hand-held Nunc-Immuno™ 12-plate washer.
Membrane-removal device.
ELISPOT plate reader QHub.

ELISA Assay

Commercially available ready-to-use Quantikine ELISA Kit to study secretion of human IFN-γ. Each kit includes a 96-well plate precoated with capture antibody, a conjugate solution, standard, diluents to perform the assay, wash buffer, color reagents (substrate solution), and stop solution.
Hand-held Nunc-Immuno™ 12-plate washer.
Centrifuge capable of spinning 15-mL culture tubes at 500 × g.
ELISA microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 or 570 nm.

Methods of ELISPOT and ELISA to Single Cells' Cytokine

Isolation of Human PBMCs

Collect blood samples from healthy donors in standard citrate-phosphate-dextrose unit bags.
Separate PBMCs using density centrifugation (500 × g for 30 min) by layering 25 mL of blood on 20 mL of 1.077 g/mL Ficoll-Paque Plus at 25°C (see Note 3).
Discard the upper plasma layer after centrifugation and transfer the blood cells into two sterile 50-mL tubes.
Resuspend the blood cells in 45 mL of sterile PBS and centrifuge for 5 min at 500 × g.
Discard the supernatant, and resuspend the pellet in 10 mL of red blood cell lysing solution and incubate for 5 min at room temperature.
After lysing, resuspend PBMCs by adding sterile PBS up to the 50-mL graduation mark on the tube.
Centrifuge the tubes for 5 min at 500 × g.
Discard supernatants and add 30–40 mL of RPMI complete medium to the tubes with PBMCs.
Mix cells 1:2 with Trypan blue dye and pipette 10 μL of that mixture into each side of a hemacytometer under a coverslip (see Note 4). Count the cells under the microscope using a 20× lens and a phase-contrast condenser.
Isolated PBMCs are divided into two pools and cells from one pool are cultured in a regular ELISPOT plate, whereas cells from the other pool are cultured in an uncoated, blank, ELISPOT plate.

ELISPOT Assay

Cells are plated into both coated ELISPOT plates and plates not coated with capture anti-IFN gamma antibodies.

Prepare uncoated ELISPOT plates and culture PBMCs as follows:

Block ELISPOT plates by adding 100 ML of block buffer per well and incubate for 1.5–5 h at room temperature.
Discard block buffer and fill each well with 200 ML of RPMI complete culture media. Allow to sit a minimum of 20 min and discard the culture media just before plating PBMCs.

Plate PBMCs (100 μL/well; six wells per group) into both uncoated and coated ELISPOT plates at cell concentrations of 10⁴ and 10⁵ cells/mL (see Notes 5 and 6).
Stimulate PBMCs with a mixture of CaI and PMA by adding them directly to the ELISPOT plate, so their final concentrations reach 0.5 Mg/mL and 50 ng/mL, respectively.
Incubate in a CO₂ incubator at 37°C for 18 h (see Notes 7 and 8).
After incubation, collect the supernatant mixture with a multichannel pipette from both uncoated and coated plates for subsequent ELISA analysis. (Uncoated plates can be discarded at this time, but coated plates are processed to develop spots.) Then, wash the plate by rinsing wells four times with wash buffer to remove PBMCs from the plate (see Notes 9 and 10).
Prepare working solutions of detection antibodies by mixing concentrated detection antibodies 1:120 with dilution buffer.
Add 100 μL of detection antibody working solution into each well and incubate ELISPOT plates overnight at 2–8°C.
Wash plates three times with the wash buffer.
Prepare a working solution of streptavidin–alkaline phosphatase by mixing the concentrated stock solution 1:120 with corre-sponding dilution buffer.
Add 100 μL of streptavidin–alkaline phosphatase working solution into each well and incubate for 2 h at room temperature.
Wash plates three times with wash buffer.
Add 100 μL of ready-to-use BCIP/NBT substrate into each well and incubate for 30–60 min at room temperature in a place protected from direct light.
Wash plates three times with distilled water and let them dry completely (see Note 11).
Quantify spots using an automated ELISPOT reader.

ELISA Assay

To remove contaminating PBMCs, centrifuge supernatants collected from coated and uncoated ELISPOT plates at 500 × g.
Pipette 500 mL of Calibrator Diluent RD6-21 into polypropylene tubes. Use the kit standards stock solution to prepare a dilution series. Mix each tube thoroughly before the next transfer. The undiluted standard serves as the high standard (1,000 pg/mL). Calibrator Diluent RD6-21 serves as the zero standard (0 pg/mL).
Add 100 μL of Assay Diluent RD1-51 to each well. Add 100 μL of standard or sample to each well (see Note 12). Cover with an adhesive strip and incubate for 2 h at room temperature.
Aspirate and wash the plate four times with wash buffer (see Note 10).
Add 200 μL of conjugate to each well. Cover with an adhesive strip and incubate for 2 h at room temperature.
Aspirate and wash the plate four times with wash buffer (see Note 10).
Add 200 μL of substrate solution to each well. Incubate in the dark for 30 min at room temperature.
Add 50 μL of stop solution to each well. The color should change from blue to yellow (see Note 13).
Determine the optical density of each well within 30 min using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 or 570 nm (see Note 14). If wavelength correction is not available, subtract readings at 540 or 570 nm from the readings at 450 nm. This subtraction corrects for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Calculation of the Amount of Cytokine per Spot (See Note 15)

Calculate the average amount of IFN gamma in the culture media collected from the uncoated plate and from the coated ELISPOT plate measured by ELISA.
Calculate the average number of spots counted with an ELISPOT reader.
Subtract the average amount of unbound (UB) IFN gamma from the total (T) amount of IFN gamma per well (0.1mL) and divide it by the average number of spots (N) to calculate the average amount of IFN gamma per each spot (C spot):

Single Cells' Cytokine Secretion Measurement by ELISPOT and ELISA Protocol

This simple protocol allows for the calculation of the average amount of IFN gamma secreted by a single cell that can be easily adapted for measuring other cytokines in a variety of species.

Although this approach has some limitations, it can be quite useful for estimating the extent of cytokine production at a single-cell level. This protocol can be easily adapted for measuring other cytokines and used for both a basic research as well as for developing diagnostic applications.

Notes of ELISPOT and ELISA to Single Cells' Cytokine

Sterilize RPMI complete culture medium and reagents that are used to separate out the white blood cells by filtering through 0.2-μm sterile filter to allow their long-term storage.
When using fetal calf serum, it is important to heat inactivate the serum at 56°C for 30 min. After the heat inactivation, the serum should be filtered.
When layering Ficoll, make sure that the blood does not mix with the Ficoll to gain the best separation and highest yield of PBMCs.
Overfilling the hemacytometer with cell solution may result in inaccurate cell quantification. While counting cells on a hemacytometer, first find the middle square which contains 25 smaller squares and count cells in 5 of them. Calculate the average and multiply by 25 (total number of squares in that area), then multiply by 2 (cell dilution factor), and multiply by 10,000 to determine the number of cells in 1 mL of original cell suspension. The resulting number should be used for calculating serial dilutions of PBMCs.
Making serial dilutions of cells allows the user to avoid overdevelopment of the ELISPOT plate and to obtain a quantifiable number of spots that can be counted either manually or using an automated ELISPOT plate reader.
For better well-to-well reproducibility, cells need to be mixed thoroughly before adding them into the wells. This may require shaking the tube containing the cells after filling every four wells in the ELISPOT plates.
Plates can be wrapped in aluminum foil to provide even heat distribution across the bottom of the ELISPOT plates during their incubation. This helps to improve well-to-well spot consistency across the plate. Aluminum foil also helps to reduce a background staining. This is a very simple procedure which can be done as follows: before plating cells, the ELISPOT plate is placed onto 13 × 16-cm piece of aluminum foil; the cells are then added into the wells, the plate is covered with the lid, and the edges of the foil are shaped loosely around the edges of the plate to wrap it. After incubating the cells, the foil can be removed and either discarded or saved and used on the next ELISPOT plate.
Shelves in the CO₂ incubator must be leveled to avoid moving cells toward one side of the well: this may produce under- and overdeveloped parts of the well and hinder quantification of spots. It is also important to avoid disturbing cultured cells (e.g., by slamming the door of the incubator) during the incubation which may cause developing weakly stained fuzzy spots.
Make sure that the height of the prongs in the handheld plate washer is properly adjusted so that prongs do not touch the membranes on bottom of the ELISPOT plates. PVDF membranes on the ELISPOT plate are fragile and are easily punctured by protruding prongs.
Between washes, it is important to tap out the excess liquid in the well onto a paper towel to prevent diluting the subsequent reagents added into the plate.
ELISPOT plates must be completely dried before analysis because wet membranes appear dark and obscure detection and quantification of spots.
To ensure accurate results, reagent addition should be uninterrupted and completed within 15 min.
If the color in the well changes to green instead of yellow or does not appear to be uniform, gently tapping the plate ensures thorough mixing.
If wavelength correction is not available, subtract readings at 540 or 570 nm from the readings at 450 nm. This subtraction corrects for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
The amount of cytokine calculated per spot is affected by cells with strong secretory activity (producing large-size spots of high-intensity staining). Therefore, for better accuracy, it may be necessary to add to this equation both (1) sizes of spots and (2) their staining intensities.

References

Alexander E. Kalyuzhny. Handbook of ELISPOT. Methods in Molecular Biology. 2012, 792. Electronic. ISSN: 978-1-61779-325-7.
Naresh Sachdeva, et al.; Cytokine quantitation: Technologies and applications. Frontiers in Bioscience. 2007, 12(12):4682-95

Reagent Solutions