Siinfekl antibody

In vitro differentiated BMDCs were collected on day 6 and seeded into 6-well plates for transduction. On day 7 and day 8, BMDCs were transduced with viruses encoding LiSmore or the control vector, or left untransduced. LiSmore-transduced BMDCs were harvested on day 10 for in vitro experiments. To assess BMDC maturation, the cells were subjected to flow cytometry analysis after staining with the following antibodies: Percp5.5-CD11c (N418), PE-H2Kb (28-14-8), APC-IA/IE (M5/114.15.2), APC-CD86 (GL-1), PE-CD80 (16-10A1), APC-CD40 (3/23), and PE-CCR7 (4B12). For the cross-presentation assay, B16-OVA cells were treated with mitomycin C (50 μg/ml) and co-cultured with BMDCs at a 1:1 ratio, either with or without pulsed blue light stimulation overnight (470 nm, 20 s ON, 5 min OFF, 1 mW/cm²). In selected groups, non-transduced BMDCs were treated with or without 2’3’-cGAMP (2 μg/ml) under the same timeframe and conditions. After 24 h, OVAp presented with MHC-I on the cell surface was detected using APC-conjugated anti-mouse H-2Kb bound to the SIINFEKL antibody (25-D1.16, BioLegend).

After cell internalization, OVA antigen would be enzymatically degraded into the functional epitopes. OVA H-2Kb-restricted CTL epitope (OVA_257−264, SIINFEKL) would interact with MHC class I complexes, leading to the presentation of the MHC class I-functional epitope (like SIINFEKL) complex on the surface of DCs. To do this assay, DC 2.4 cells were cultured in 96 well cell culture plates, and pulsed with OVA in complex with LDH in RPMI 1640 medium (without GM-CSF) for 16 h. Then cells were harvested and washed, and stained with APC or PE anti-mouse H-2Kb of MHC class I bound to SIINFEKL antibody (Clone 25-D1.16; BioLegend) to determine the degree of epitope presentation (SIINFEKL/H-2Kb complexes) on DC2.4 cell surface.