HumanIL-2ElISPOTKit.docVIP

Human IL2 Elispot Kit for Human IL2 Elispot Kit Reagents for 20 x 96 Well Plates Materials Required: 96 PVDF-bottomed-well plates if not ordered . Cell culture media CO2 incubator 70% ethanol Tween 20 Phosphate buffered saline Assay Control: IL2 production by PBMC upon stimulation by PMA and Ionomycin. This protocol is given as a suggestionDilute PBMC in culture media e.g. RPMI 1640 supplemented with 2mM L-glutamine and 10% heat inactivated fetal calf serum containing 1ng/ml PMA and 500ng/ml ionomycin Sigma, Saint Louis, MO . Distribute 5 x 104 to 1 x 105 cells in antibody coated PVDF-bottomed-wells and incubate for 15-20 hours in an incubator. For other stimulators incubation times may vary, depending on the frequency of cytokine producing cells, and should be optimised in each situation. Reagent Preparation: Detection antibodyReconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.If not used within a short period of time, reconstituted detection antibody should be aliquoted and stored at -20°C. In these conditions the reagent is stable for at least one year. Streptavidin alkaline phosphatase:Dilute 1/5000 in PBS 1% BSA. We recommend to proceed in two steps: Step 1: Pre-dilution 1/100: 5μl of streptavidin alkaline phosphatase in 495μl of PBS 1% BSA. Step 2: Dilution 1/50: For one plate 200μl of pre-dilution + 9.8 ml of PBS 1% BSA. PREPARATION IMMEDIATELY BEFORE USE IS RECOMMENDED. Phosphate buffered saline 10X Concentrate solution .For 1 liter weigh: 80g NaCl; 2g KH2PO4; 14.4g Na2HPO42H2O. Add distilled water to 1 liter. Check that pH is 7.4 +/- 0.1. Dilute the solution to 1X before use. 2% dry skimmed milk in PBSFor one plate dissolve 0.2 g of powder in 10 mL of 1X diluted PBS. 1% BSA in PBSFor one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS. 0.1% Tween in PBSFor one plate dissolve 100μl of Tween 20 in 100 ml of 1X dilu