TLR blocking assay

ZY Zhen Yang
YZ Yiwen Zhang
QZ Qin Zhao
SD Senyan Du
XH Xiaobo Huang
RW Rui Wu
QY Qigui Yan
XH Xinfeng Han
YW Yiping Wen
SC San-Jie Cao
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To determine the pattern recognition receptors for HbpA on 3D4/21 macrophages, we blocked toll-like receptors 2 (TLR2) and 4 (TLR4) using Ultra-LEAF™-purified CD282 (TLR2) (BioLegend, Cat: 153,001) and LEAF™-purified anti-mouse TLR4 (CD284)/MD2 (BioLegend, Cat: 117617). Each antibody was used at a final concentration of 8 µg/mL. 3D4/21 cells were plated into 6-well plates at a density of 1 × 106 cells/well, cultured overnight, incubated for 2 h with a single antibody, and then incubated overnight with 20 μg/mL rHbpA protein. Cell culture supernatants were collected for detection of secreted cytokines using ELISA kits as described in “Detection of proinflammatory cytokines using ELISA and qRT-PCR” section.

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