Apc conjugated anti cd11b antibody

Whole blood obtained cardiac puncture was buffered with EDTA, subjected to red-cell lysis using BD FACS Lysing Solution (BD Biosciences), and washed in PBS supplemented with 3% fetal calf serum. Blood leukocytes were first incubated with rat anti-mouse FcR/III antibody (2.4G2) (BD Biosciences) to minimize non-specific binding of antibody to FcR. They were further incubated with APC-conjugated anti-CD11b antibody (BD Biosciences) and PE-conjugated anti-F4/80 antibody (BD Biosciences) for 10 min on ice, and washed with PBS supplemented with 3% fetal calf serum. The percentages of CD11b + and F4/80 + cells were analyzed by the FACS Canto II flow cytometer (BD Biosciences) using EXPO32 software (Beckman Coulter).

To quantitatively assess cell internalization, Sen or Res cells were labelled with 3 μM CFSE (carboxyfluorescein diacetate succinimidyl ester) dye (Stemcell Technologies, VIC, Australia) for 10 min at 37⁰ C in serum-free RPMI 1640. Labelling was stopped with complete media and the cells were washed twice prior to co-culture with macrophages. 5×10⁴ THP-1 macrophages were co-cultured (ratio of 1:1) with the CFSE labelled Sen or Res or D3 cells. 50 μg of Res-MP or Sen-MP or D3-MPs were added to the heterotypic cell cultures and following 24 h incubation cells were harvested and stained with a macrophage marker, APC conjugated anti-CD11b antibody (BD Biosciences). Samples were incubated for 30 min in the dark, washed twice in PBS and analysed for dual labels and single labels with the BD LSR Fortessa™ X-20 flow cytometer. The cells which were dual positive for both markers (CFSE-green channel and CD11b-red channel) represent those cells engulfed by macrophages. The remainder of the population comprises of macrophages alone, cells which have engulfed macrophages or cells alone. This was measured by the percentage drop in the population of each of these in their respective channels.