U bottom 96 wells plate

TLR4 and CD14 endocytosis was quantified by flow cytometry as described previously (Perkins et al., 2018 (link)). Briefly, 1 × 10⁶ viable BMDMs were incubated with LPS for the indicated time points (37°C, 5% CO₂) at a volume of 2 mL in sterile flow cytometry polypropylene round-bottom tubes (Corning). After incubation, 2 mL of ice-cold FACS Buffer (PBS containing 0.5% FBS and 2 mM EDTA) was added, the cells were centrifuged (400 × G, 5 min, 4°C), and washed twice in FACS Buffer. The cells were then resuspended in 100 μL of FACS Buffer containing 20 μg mL⁻¹ anti-CD16/32 (Fcγ RII-Blocking antibody) (BioLegend, 101302), transferred to a 96-wells U-bottom plate (Corning) and incubated for 20 min on ice. The plates were then centrifuged (500 × G, 5 min, 4°C), the supernatant discarded, and the cells resuspended in 100 μL of FACS Buffer containing 4 μg mL⁻¹ PE-conjugated anti-TLR-4 (BioLegend, 145403), 1 μg mL⁻¹ APC-conjugated anti-CD14 (BioLegend, 123311) or 100 μL of FACS Buffer containing 4 μg mL⁻¹ PE-conjugated IgG2a κ-chain isotype-matched control (BioLegend, 400507) and incubated on ice for 30 min in the dark. After three washes, the samples were then resuspended in FACS Buffer and read on a LSR II flow cytometer (BD). Analysis were done on FlowJo v10 (BD), and percentage of surface TLR4 was calculated as follows: (MFI_t=x – MFI_{isotype control})/(MFI_t=0 – MFI_{isotype control}).