Pe conjugated antimouse cd4

Flow cytometry analysis was performed as previously described 3 using the following antibodies: APC‐conjugated antimouse F4/80 (eBioscience, San Diego, CA, USA), APC‐conjugated antimouse CD16/32 (Biolegend, San Diego, CA, USA), APC‐conjugated antimouse CD206 (Biolegend), PE‐conjugated antimouse Dectin‐1 (Biolegend), PE‐conjugated antimouse CD4 (eBioscience) and FITC‐conjugated antimouse CD69 (eBioscience).

The CD8⁺ T cell hybridoma cell line B3Z, the melanoma cell line B16.F10, and H-2^b fibroblast cell line K41 were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (GIBCO, NY, USA) and 100 U/ml streptomycin/penicillin. Raw264.7 cells and HEK293 cells (a cell line that does not express TLR2/TLR4) [17 (link)] were maintained in complete DMEM (GIBCO) supplemented with 10% fetal calf serum (FCS) and 100 U/ml streptomycin/penicillin. The cells were maintained at 37°C in an atmosphere containing 5% CO₂.
The HBc_87–95 (SYVNTNMGL), OVA8 (NH2-SIINFEKL-COOH), and OVA20 (NH2-SGLEQLESIINFEKLTEWTS-COOH) peptides were synthesized by GL Biochem Ltd. (Shanghai, China) to >95% purity. Soluble PE-HBc_87-95 tetramers were synthesized by QuantoBio (Beijing, China). The following antibodies were obtained as indicated: anti-grp94, anti-His tag, anti-TLR2, anti-TLR4, anti-pIκB-α, anti-β-actin and anti-p65 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). PerCP-Cy5.5-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD8, PE-conjugated anti-mouse CD4, and APC-conjugated anti-mouse IFN-γ antibodies were from eBioscience (San Diego, CA, USA), APC-conjugated anti-human CD11b antibody was from Biolegend (San Diego, CA, USA).

17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (DMAG) was purchased from Invivogen and NVP‐AUY922 (AUY) were bought from Selleckchem. Primary monoclonal (mAb) antibodies were obtained from the following sources: Purified anti‐mouse CD3, Alexa647‐conjugated anti‐mouse CD4, PerCP‐ or eFluor 450‐conjugated anti‐mouse CD8, FITC (fluorescein isothiocyanate)‐conjugated anti‐mouse I‐A^b, biotin‐conjugated anti‐mouse CD90.1, PE‐conjugated anti‐mouse/human Helios, APC (Allophycocyanine)‐conjugated Annexin V: Biolegend (London, UK); Purified anti‐mouse CD95, APC‐conjugated anti‐mouse CD107a, PE‐conjugated anti‐mouse Granzyme B, biotin‐conjugated anti‐mouse I‐A^b; Purified anti‐mouse TCR‐β: BD biosciences (Heidelberg, Germany); PE‐conjugated anti‐mouse CD4, eFluor 450‐conjugated anti‐mouse CD8, PE‐Cy5‐conjugated anti‐mouse Foxp3: eBiosciences (Frankfurt/Main, Germany).