ELISPOT Protocol
实验概要
The Elispot (Enzyme Linked Immuno-Spot) assay provides an effective method of measuring the antibody or cytokine production of immune cells on the single cell level. The popularity of this assay has seen resurgence in recent years as researchers attempt to gain a better understanding of immune responses in a variety of applications. The following protocol is an example of a typical Elispot assay for quantifying the number cells producing interferon- (IFN-y) in response to antigen or non-specific activation using phytohemagglutinin (PHA). It may be optimized as necessary for other applications.
主要试剂
Phosphate Buffered Saline (PBS):
80.0 g NaCl
14.4 g Na2HPO4
2.4 g KH2PO4
2.0 g KCl
Add ddH2O up to 10 L; pH to 7.2 with HCl
Coating Buffer:
Can use either Sterile PBS or
Sterile Carbonate Buffer (per ELISA protocol)
8.4 g NaHCO3
3.56 g Na2CO3
Add ddH2O up to 1.0 L, pH to 9.5.
PBS-Tween:
0.05% Tween-20 in PBS
(500 μl Tween-20 in 1L PBS)
Blocking Buffer (PBS-BSA):
1% BSA in PBS
(10 g BSA-Fraction V in 1L PBS)
PBS-Tween-BSA:
1% BSA in PBS-Tween
(10 g BSA-Fraction V in 1L PBS-Tween)
Tissue Culture (TC) Medium:
As appropriate for cells being analyzed
AEC Solution:
100 mg AEC (3-amino-9-ethyl-carbazole) in 10 ml DMF (N,N, Dimethylformamide)
Solution should be prepared in a glass tube in a fume hood.
AEC Buffer:
(0.1 M Acetate): 148 ml 0.2 M acetic acid (11.55 ml glacial acetic acid per liter of water) and 352 ml of 0.2 M sodium acetate (27.2 g per liter of water)
Bring up to 1L with water and adjust to pH 5.0 if required
Substrate Solution:
800 μl AEC solution in 24 ml AEC buffer
Filter with 0.45 μm filter and add 12 μl 30% H2O2
Use immediately
实验步骤
1. Coat the Plate:
1) Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s LEAF™ format antibodies are specifically designed for this assay) to a final concentration of 0.5–4 µg/ml in sterile Coating Buffer and transfer 100µl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore; Cat. No. MAIPS-4510).
2) Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in humidified atmosphere.
2. Block the Plate:
1) Wash plate 3 times with sterile PBS, 200 µl/well.
2) Add 200 µl/well of sterile Blocking Buffer.
3) Seal plate and incubate at room temperature for ≥ 1 hour.
4) Wash plate 3 times with sterile PBS, 200 µl/well.
3. Set-Up Tissue Culture and Add Antigen or Mitogen:
1) Add appropriate sterile antigen or mitogen solution diluted in appropriate sterile tissue culture medium (TC) to ELISPOT plate, 100 µl/well.
2) Add cells diluted in sterile TC medium, 100 µl/well. Use 50,000-500,000 cells/ well (the minimum number of cells should be determined in preliminary experiments).
3) Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the optimum stimulation period. BioLegend recommends a 24 hour incubation for IFN-γ, IL-2, and TNF-α, and a 48 hour incubation period for IL-4, IL-5, and IL-10 for most activation conditions.
4. Add Detection Antibody:
1) Wash plate 3 times with PBS, 200 µl/well.
2) Wash plate 3 times with PBS-Tween, 200 µl/well.
3) Add 100 µl/well of diluted biotinylated detection antibody at 0.25-2 µg/ml in PBS-Tween-BSA.
4) Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature.
5. Add Avidin-Horseradish Peroxidase (Av-HRP):
1) Wash plate 4 times with PBS-Tween, 200 µl/well.
2) Add 100 µl per well of the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate diluted to its pre-determined optimal concentration in PBS-Tween-BSA (usually between 1/500 – 1/2000).
3) Seal the plate and incubate at room temperature for 1 – 2 hours.
4) Wash plate 3 times with PBS-Tween, 200 µl/well.
5) Wash plate 3 times with PBS, 200 µl/well.
6. Add Substrate:
1) Add 200 µl/well of fresh Substrate solution.
2) Monitor spot/color development at room temperature and stop reaction by rinsing plate with tap water and vigorously flicking plate over a waste container or sink, followed by blotting on paper towels or other absorbent materials.
3) Air dry plate overnight, until it is completely dry.
4) Count spots manually with a dissecting microscope or using an automated image acquisition/analysis unit (plates can be analyzed for up to 3 months).
