CD14 Monoclonal Antibody (Sa2-8), FITC, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Immortal macrophages, primary bone marrow derived macrophages and DCs or splenic DCs (0.5×106) of the indicated genotypes were treated with E.coli LPS and/or oxPAPC for the indicated time points at 37°C.	Cells were then washed with 1 mL cold PBS and stained for appropriate antibodies on ice for 20 to 30 min. 2% mouse serum or rat serum were used as the blocking reagent to reduce non-specific binding of the antibodies. The stained cells were then washed with 1ml cold PBS and resuspended in 200 μL PBS.	Staining of the surface receptors was analyzed with BD FACSCanto II.

Upstream tips
Immortal macrophages, primary bone marrow derived macrophages and DCs or splenic DCs (0.5×106) of the indicated genotypes were treated with E.coli LPS and/or oxPAPC for the indicated time points at 37°C.
Protocol tips
Cells were then washed with 1 mL cold PBS and stained for appropriate antibodies on ice for 20 to 30 min. 2% mouse serum or rat serum were used as the blocking reagent to reduce non-specific binding of the antibodies. The stained cells were then washed with 1ml cold PBS and resuspended in 200 μL PBS.
Downstream tips
Staining of the surface receptors was analyzed with BD FACSCanto II.

Publication protocol

Immortal macrophages, primary bone marrow derived macrophages and DCs or splenic DCs (0.5×106) of the indicated genotypes were treated with E.coli LPS and/or oxPAPC for the indicated time points at 37°C. Cells were then washed with 1 mL cold PBS and stained for appropriate antibodies on ice for 20 to 30 min. 2% mouse serum or rat serum were used as the blocking reagent to reduce non-specific binding of the antibodies. The stained cells were then washed with 1ml cold PBS and resuspended in 200 μL PBS. Staining of the surface receptors was analyzed with BD FACSCanto II. The mean fluorescence intensity (MFI) of CD14 and TLR4 from unstimulated or stimulated cells were recorded. The percentage of surface receptor staining at indicated time points, which is the ratio of the MFI values measured from the stimulated cells to those measured from the unstimulated cells, was plotted to reflect the efficiency of receptor endocytosis. For measuring the extent of TLR4/MD-2 dimerization, the percentage of TLR4/MD2 dimer was calculated by 100%-the percentage of TLR4/MD-2 monomer. The percentage of the TLR4/MD-2 monomer was determined by the ratio of the MFI values (obtained from MTS510 antibody staining) of the stimulated cells to those of the unstimulated cells.

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