Pe anti tlr 4

PE-Mφ from MGL1^−/− and WT mice were obtained and stimulated with TcAg (25 µg/mL) for 24 or 48 h. These cells were incubated with anti-mouse FcγR antibody (CD16/CD32) in staining buffer (1× PBS, 2% FBS, 1% NaN3) for 15 m, followed by incubation for 30 m at 4 °C with FITC-conjugated anti-F4/80, PE-conjugated anti-MGL1 and APC-conjugated anti-MGL2 antibodies (BioLegend, SD, CA). For quantification of costimulatory molecule expression, MGL1^−/− and WT PE-Mφ and BMMφ were stimulated in vitro with LPS (100 ng/mL) or TcAg (25 µg/mL) for 24 h or infected with culture-derived epimastigotes for 2 h (ratio 1:10). The cells were incubated with the following fluorochrome-conjugated Abs: Pacific blue anti-F4/80, PerCP/Cy5.5 anti-CD11b, PE anti-TLR-4, PE anti-MHC-II, FITC anti-TLR-2, FITC anti-CD40 and FITC anti-CD80 (all from BioLegend), as well as the negative control. Mφ were washed three times with FACS buffer and fixed in 0.8% paraformaldehyde before acquisition and analysis (Attune NxT, Thermo Fisher Scientific, Waltham, MA, USA).

The following fluorophore-conjugated antibodies were used: PE anti-TLR4 (Biolegend; clone Sa15-21), PE anti-TLR4/MD2 (BD; clone MTS510). Anti-mouse CD16/CD32 (BD; mouse Fc blocker, clone 2.4G2) were used as the blocking reagent to reduce the non-specific binding of the antibodies. Stained cells were read with flow cytometry (LSRII; BD Biosciences, San Jose, CA, USA), and the result was analyzed by FlowJo software (Flow Jo LLC, Ashland, OR, USA).
The efficiency of TLR4 endocytosis was calculated as the ratio of the MFI values of anti-TLR4 (clone Sa15-21) measured from the stimulated cells to those from the unstimulated cells. The extent of TLR4 monomerization was determined by the ratio of the anti-TLR4/MD2 MFI values (clone MTS510) of the stimulated cells to those of the unstimulated cells. For intracellular staining of TLR4/MD2, Cytofix/Cytoperm solution (BD) was added prior to fixation and permeabilization.