H 2kd pe

The mouse anti-CHIKV-nsP2 monoclonal antibody, used in this study was developed by us [58 (link)]. The anti-CHIKV-E2 monoclonal antibody was a kind gift from Dr. Manmohan Parida, DRDE, Gwalior, India. HRP linked secondary antibodies, H-2k^d PE, I-A/I-E PE, isotype PE, isotype APC and HSP90 antibodies were purchased from BD Biosciences (San Jose, CA, USA). CD86 APC and CD80 APC were purchased from eBiosciences (San Diego, CA, USA). The monoclocal antibodies for cleaved caspase-3 (Asp175), cleaved caspase-8 (Asp387) and caspase-9 (C9) were purchased from cell signaling technology (Danvers, MA, USA). The anti-mouse Alexa Fluor 488 was purchased from Invitrogen (Carlsbad, CA, USA). Mouse IgG1 isotype control, GAPDH and β-actin were purchased from Abgenex India Pvt. Ltd. (Bhubaneswar, India). Saponin, Anisomycin and Bovine serum albumin fraction V were purchased from Sigma-Aldrich. 17-Allylaminogeldanamycin (17-AAG) and Z-VAD-FMK were purchased from Merck Millipore (Billerica, MA, USA).

We purchased directly conjugated monoclonal antibodies for flow cytometry from Invitrogen (Waltham, MA, USA): CD4-PE, Clone GK1.5; CD11b-ef450, Clone M1/70; CD44-FITC, Clone IM7; CD45AF700, Clone 30-F11; CD206-APC, Clone MR6F3; FoxP3-ef450, Clone FJK-16S, IFN-γ-PE, Clone XMG1.2; GzmB-APC, Clone GB11; iNOS-PE, Clone CXNFT; live dead fixable dead cell stain kit, Catalogue No. L34959. BD Pharmingen (San Jose, CA, USA): CD3-AF700, Clone 17A2; CD4-Pacblue and perCPcy5.5, Clone RM4-5; CD8-FITC, perCPcy5.5, and PECY7, Clone 53-6.7; CD62L-PECY7, Clone MEL-14; H2Kd-PE, Clone 17A2; Ly6G-PE and percpcy5.5, Clone 1A8; Ly6C-APCCY7, Clone- AL-21; TNF-α PECY7, Clone MP6-XT22. Biolegend (San Diego, CA, USA): CD3-PECY7 Clone 17A2; B220-percpcy5.5, Clone RA3-6B2. R&D Systems: CCR2-APC, Clone 475301; CCL2-APC, Clone 123616. eBioscience (San Diego, CA, USA): H2Kd/Dd-ef450 Clone 34-1-25; CD4-APC, Clone GK1.5.

Immunostaining was performed as described previously.^{41 (link)} Briefly, after 10 min of incubation at 4 °C with Fc receptor blocker (BD Biosciences, San Joses, CA, USA), the cells were stained with the corresponding antibodies. The antibodies used for DC phenotyping were CD11c-PE, CD14-FITC, CD40-PE, CD54-FITC, CD80-PE, CD86-FITC, H-2K[d]-PE, and I-A[d]-FITC; all these antibodies were purchased from BD Biosciences. For intracellular cytokine staining, the cells were incubated with brefeldin A (BD Biosciences) for 1 h. The cells were stained with surface markers, such as CD4-FITC and CD25-APC (BD Biosciences). Then, the cells were permeabilized using Cytofix/Cytoperm reagents (BD Biosciences) and stained for intracellular markers as follows: IFN-γ-PE, IL-4-PE (BD Biosciences), IL-17A-PE, and Foxp3-PE (eBioscience, San Diego, CA, USA). Stained cells were acquired using a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using the FlowJo software (v.10, Ashland, OR, USA).