Surface staining was used to detect the upregulation of cell surface markers on DCs (DC2.4 cells or BMDC) and splenocytes in response to the stimulants. Intracellular cytokine staining (ICS) was used to detect cytokine production in response to in vitro stimulation. After defined incubation periods (24 h for surface staining and 4 h for ICS) with stimulants at an MOI (Multiplicity of Infection) of 1:10, DCs were washed twice with 0.5% BSA supplemented PBS (PBSA). Surface staining was performed using monoclonal anti-mouse antibodies such as Phycoerythrin (PE) labeled murine CD11c (clone N418), Allophycocyanin (APC) labeled CD40 (clone 3/23), APC labeled CD80 (clone 16-10A1), Alexa Fluor® 700 labeled CD86 (clone GL1), eFluor® 450 labeled MHCII (clone AF6-120.1). Surface stained cells were fixed using 2% paraformaldehyde and permeabilized with Perm/wash buffer. Monoclonal anti-mouse antibodies against proinflammatory cytokine TNF, Alexa Fluor® 700 labeled TNF (clone MP6-XT22) were used for staining the permeabilized DCs. Stained DCs were further washed and re-suspended in PBSA and stored at 4°C in dark until flow cytometry analysis.

In case of in vitro restimulation of splenocytes, Brefeldin A was added at 1/1000 dilution during the final 6 h of incubation. After 24 h of incubation, splenocytes were washed twice with PBSA and surface stained in the presence of Fc block (clone 2.4G2) with monoclonal anti mouse antibodies Pacific Blue™ labeled CD4 (clone RM4-5) and FITC labeled CD8a (clone 53–6.7). Following staining, splenocytes were fixed and permeabilized with Cytofix/Perm buffer. Monoclonal anti mouse antibodies Alexa Fluor® 647 labeled IFNγ (clone XMG1.2) and PE labeled TNF (clone MP6-XT22) were used to stain the permeabilized splenocytes for detecting the cytokines. Stained cells were finally washed and resuspended in PBSA and stored at 4°C in the dark. For all the in vitro stimulation assays, unstimulated and unstained cells were maintained as controls. All flow cytometry reagents and antibodies were purchased from BD Pharmingen, San Jose, CA. Multi-parameter flow cytometry was performed using BD FACSAria™II (BD, Franklin Lakes, NJ) and data acquired with BD FACSDiva™ software (BD, Franklin Lakes, NJ). Flow cytometric data analysis was carried out with FlowJo software (Tree Star, San Carlos, CA).

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