MouseELISPOT-Assay.docVIP

本文章来源于SciMall科学在线 Mouse ELISPOT-Assay To check frequency of cytokine IFN-γ , IL-5, IL-2, IL-10, IL-4 positive cells following in vitro Ag-specific activation. 1. Set up 96-well plate map 2. Pre-coating of ELISPOT 96-well plates with primary antibodies: - new ?white“ plates from Whatman/Polyfiltronics, or white plastic nitrocellulose plates from Millipore - 100μl of sterile PBS with - anti-mIFN-γ R46A2 hybridoma or Pharmingen , final 4μg/ml - anti-mIL-2 Pharmingen , final 2μg/ml - anti-mIL-4 11B11-hybridoma or Pharmingen , final 4μg/ml - anti-mIL-5 TRFK-5-hybridoma or Pharmingen , final 5μg/ml - anti-mIL-10 not tested yet – Pharmingen , final ? μg/ml - incubate o/n, 4° humidified chamber Tupper, with soaked paper towels C, 3. Before getting the mice preparation of: Collection of blood/serum samples Bleeding of mice by cardiac puncture: prepare Halothane glass cylinder, cotton, syringes, needles, and eppendorf vials labeled. cHL-1 medium; calculation of volume: a proliferation assay # organs x # antigens x # replicates x 0.1ml b cytokine supernat. # organs x # antigens x # replicates x 0.5ml c ELISPOT # cytokines x # organs x # antigens x # replicates x 0.1ml x # of cell suspensions d spleen; 107/ml final cell concentration 106/well e LN; 5x106/ml Add L-glutamin, and Pen/Strep, sterile filter. when not testing ex vivo peptide response, one can also successfully use RPMI or DMEM, 10% FCS; with other antigens! 1 continue preparing ELISPOT plates - wash plates 3x with sterile PBS 200μl - block 1hr with sterile PBS with ?highgrade“ cell culture, BSA fraction V, Sigma 1% BSA 200μl - wash 3x with sterile PBS 200μl 2 Plate out the antigens in fresh cHL-1 in ? of the final volume at 2x concentration : a medium cHL-1 b Ovalbumin: OVA final 13.5μg/ml; ?optimal“ for OVA T cell clones 2.7μg/ml c Concanavalin A final 2μg/ml from stock 100μg/ml stock in this case with Balb/c for relevant I-A and irrelevant I-A spleen APC or