Ctl immunospot s4 analyzer

GrzB ELISpot’s were performed in accordance with manufacturer instructions (Mabtech). 1×10⁵ memory CD4 and memory CD8 T cells were cultured (200 μl/well) for 24 hrs in 96-well flat-bottomed nitrocellulose plates (MultiScreen-HTS, Millipore). Wells were first prepared by treating with 70% ethanol for 2 mins and rinsing with sterile H₂0. Wells were then coated overnight at 4°C with 15 μg/ml GrzB mab (clone GB10). After GB10 coating, wells were washed with PBS, and pre-incubated with cell culture RPMI-1640 medium (200 μl/well) for 30 mins at room temp. Cells were then added to appropriate wells and either unstimulated (UT) or costimulated with 1 μg/ml soluble CD3 (clone CD3-2, Mabtech) + soluble CD28 mabs (clone CD28.2, BD Biosciences) for 48 hrs wrapped in aluminum foil at 37°C + 5%CO₂. After the treatment period, cells were gently harvested for flow cytometry staining, and wells were washed with PBS. 1 μg/ml biotinylated GrzB detection mabs (clone GB11) were added for 2 hrs at room temp. Wells were washed and streptavidin-HRP added for 1 hr. Wells were washed and TMB substrate added until spot development (10-30 mins). Color development was stopped with H₂0, and plates were air-dried, wrapped, and stored at room temp. until spot analysis. Spots were analyzed using CTL-ImmunoSpot S4 analyzer (Cellular Technology Limited).