Assessing OT-1 Memory Cell Cytotoxicity

Mice harboring memory OT-1 cells, primed with MCMV-SIINFEKL or RAE-1γMCMV-SIINFEKL, were sacrificed and splenocytes isolated using a standard protocol. 2x10⁶ cells were then incubated for 6h with different concentrations of SIINFEKL peptide (JPT PeptideTechnologies GmbH) in RPMI 1640 (PAN-Biotech) supplemented with 10%FCS (PAN-Biotech), Brefeldin A (Invitrogen), Monensin (Invitrogen), and CD107 (Invitrogen) at 37°C.
For the in vitro killer assay, mice harboring memory OT-1 cells (CD45.1) were sacrificed and splenocytes isolated using a standard protocol. Splenocytes were pooled from 4-5 mice/group. CD8 T cells were purified by negative selection using magnetic beads (Miltenyi Biotec), and OT-1 (CD45.1) cells were stained with CD45.1 antibody and sorted using FACSAria II (BD) using high-speed sorting into RPMI supplemented with 20% FCS. Sorted OT-1 (CD45.1) cells were co-incubated with E.G7-OVA (CD45.2) cells for 4h in 2:1, 1:1, and 0.5:1 effector to target ratios at 37℃. After co-incubations, target cells were identified as CD45.1 negative, and viability was determined using Fixable Viability Dye (eBioscience). OT-1 cytotoxicity was calculated using following formula: [(% FVD⁺CD45.1^- cell-specific lysis − % FVD⁺CD45.1^- cell spontaneous lysis)/(100 − % FVD⁺CD45.1^- cell spontaneous lysis)] × 100 as described in (19 (link)).