Ova specific mhci dextramer apc

C57Bl6 mice received a subcutaneous graft of 1*10⁶ OVA-expressing E0771 cells and tumors were allowed to develop. De-immortalized DCs were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with either no additions (controls), NP conditions or classical activation scheme for 24 h. Cells were then pulsed with OVA protein and pooled together per conditions. Next, DCs cells were administered intravenously (2*10⁶ cells per mouse in 100 µl PBS) in the OVA-E0771 tumor-bearing mice at 3 days post tumor grafting and again one week later (10 days following original tumor grafting). Tumor growth was then evaluated using caliper measurements for a period of up to 23 days. After that time, tumors and spleens were isolated and single cell suspensions were made. For the tumor samples, CD8⁺ T cells were isolated using FACS sorting and the level of granzyme B was determined by ELISPOT, while the level of perforin was determined by ELISA. From the spleen, CD8⁺ T cells were isolated by FACS and stained with OVA-specific MHCI dextramer-APC (Immudex) diluted at 1:2 and analysed using ImageStreamX Mark II analysis.