Anti siinfekl kb apc antibody

Peptide-specific CD8⁺ T cell lines were used as read-out for the antigen presentation capacity of infected or non-infected cross-presenting BMDCs. 2 × 10⁵ CD8⁺ were co-cultured with 4 x 10⁵ BMDCs for 4 h in the presence of 1 μg/ml brefeldin A (Merck) at 37°C and 5% CO₂. T cell activation of CD8⁺ T cells was determined by cytokine production analyzed by intracellular cytokine staining (ICS) as described below. For detection of SIINFEKL/K^b complexes at the cell surface, anti-SIINFEKL/K^b APC antibody (eBioscience 25-D1.16) was used after CD16/32-Fc-blockade (2.4G2, BD) and viability dye (Invitrogen). FACS analysis was performed on BD FACS CantoII and FlowJo 6.4.2 software.

ICS (intracellular cytokine staining) was performed as described earlier (15 (link)). Briefly, cells were washed with FACS buffer and then stained with 1 μg/ml ethidium monoazide bromide (Life Technologies GmbH) on ice under bright light for 20 min to mark dead cells. Surface markers stained by anti-CD8α antibodies (APC anti-CD8α, 5H10, Invitrogen) for 30 min. Cells were then fixed and permeabilized according to the manufacturer's protocol (BD Cytofix/Cytoperm™ Kit). Cells were stained with anti-IFNg antibodies (FITC anti-IFNg, XMG1.2, BD) for 30 min. Finally, cells were fixed with 1% PFA and used for FACS analysis.
For detection of SIINFEKL/K^b complexes at the cell surface, anti-SIINFEKL/K^b APC antibody (eBioscience 25-D1.16) was used after CD16/32-Fc-blockade (2.4G2, BD) and viability dye (Invitrogen).
FACS analysis was performed on BD FACS CantoII and FlowJo 6.4.2 software.