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Monday, May 03, 2004
Description ELISPOT (Enzyme-linked ImmunoSPOT) technique was originally used to enumerate antibody secreting B cells. In the current technique, cells are deposited onto a membrane coated with one antibody specific for a protein followed by an appropriate incubation period. Subsequently, the protein of interest is detected in the environment immediately surrounding the cell secreting it, with another antibody specific for a different epitope of the protein. The signal detected by the HRP enzyme/substrate results in a colorimetric footprint of the cells and can be quantitated by visual scoring or specialized plate-readers. Procedure Materials 96 well plate (Millipore, Cat. No. MAIPS4510) Capture and detection antibodies for the cytokine of interest (please see the Reference Table above) RPMI-1640 supplemented with 10% FBS HRP-conjugated Avidin (AV-HRP, Cat. No. 18-4100) AEC substrate (Sigma, Cat. No. A-5754) Buffers Coating Buffer D-PBS, pH 7.2 eBioscience Assay Diluent Cat. No. 00-4202 Wash Buffer Substrate Solution Distilled water Instruments Pipettes and pipettors Refrigerator Incubator Plate Washer: Wash bottle or automated wash machine. ELISPOT reader (or visual inspection) Experiment Duration 1 overnight antibody incubation, 1 overnight cell activation 3-5 hour washing, antibody incubation, analyzing samples Method Coat ELISPOT plate with 100 祃/well of purified capture antibody at 10 礸/ml in Coating Buffer, incubate at 4癈 overnight. Wash 6x with Wash Buffer. Block plate with 200 祃/well of complete RPMI-1640 at room temperature for 1 hour. Seed activated cells at 100 祃/well and incubate at 37癈, 5% CO2 humidified incubator for 24 hours. Note: Cells can be diluted in a regular tissue culture plate starting at 106/well in triplicate wells with 1:3 or 1:4 serial dilution down the plate, and then transfer to the ELISPOT plate. Wash wells 5x with Wash Buffer and 1x with distilled water. Add 100 祃/well of biotinylated detection antibody at 1 礸/ml in Assay Diluent and incubate at room temperature for 2 hrs. Wash 6x with Wash Buffer. Add 100 祃/well of AV-HRP at 1/1000 dilution in Assay Diluent and incubate at room temperature for 30 minutes. Wash 6x with Wash Buffer. Add 100 祃/well of AEC Substrate Solution and develop at room temperature for 20-60 minutes until visible spots appear. Wash plate 3x with 200 祃/well distilled water. Air-dry the plate and count spots by using a dissecting microscope or ELISPOT reader. Cytokine ELISPOT Buffers ELISPOT Coating Buffer Dulbecco抯 Phosphate Buffered Saline (D-PBS): 8 g NaCl 0.2 g KCl 1.44 g Na2HPO4?H2O 0.24 g KH2PO4 Add DI H2O to 1 L. Adjust pH to 7.2. Autoclave or sterile-filter (0.22 祄) and store at 4癈. ELISPOT Diluent Buffer 1X PBS 10% FBS (Hyclone Cat. No. SH30088) ELISPOT (ELISA) Wash Buffer 1X PBS 0.05% Tween-20 AEC Substrate Solution Dissolve 4 mg of AEC in 1 ml of DMF (Dimethyl Formamide, Pierce No. 20672) Add 14 ml of 0.1 M phosphate-citrate buffer, pH 5.0 (see below for recipe) Filter through a 0.45 祄 filter. Just before use, add 10 祃 of 30% H2O2. 1X PBS 80.0 g NaCl 11.6 g Na2HPO4 2.0 g KH2PO4 2.0 g KCl DI H20 up to 10.0 l pH to 7.0 0.1 M Phosphate-Citrate-Buffer (pH 5.0) Citric acid: 9.6 g to 500 ml with DI water Dibasic sodium phosphate: 14.2 g to 500 ml with DI water Add dibasic sodium phosphate to citric acid solution until the pH = 5.0 Dilute: 1:1 with DI water Recipes Supplies Tips (责任编辑：泉水) |
Cytokine ELISPOT Protocol
时间:2006-07-24 14:10来源:Scienceboard.net 作者:admin
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