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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