Cd209 apc

After co-culture of 3DJPCs/3DOBJPCs and M1/M2 macrophages for five days, macrophages were detached by using TrypLE-Express and the expression of cell surface markers of M1 or M2 macrophages was analyzed by flow cytometry. After centrifugation (1400 rpm, 5 min) of the collected macrophage suspension and removal of the supernatant, cell pellets were incubated with 10% Gamunex (human immune globulin solution, Talecris Biotherapeutics GmbH, Frankfurt, Germany) on ice. The cells were then incubated with fluorophor labeled CD80-PE, CD86-PE, and CD209-APC antibodies (Biolegend, San Diego, CA, USA) for 30 min in the dark. Subsequently, cells were washed twice with FACS buffer (PBS containing 0.1% BSA and 0.1% sodium azide), and surface marker expression was measured using a Guava EasyCyte 6HT-2L flow cytometer (Merck Millipore, Darmstadt, Germany). For data evaluation, guavaSoft 2.7 (EMD Millipore Corporation, Hayward, CA, USA) was used.

Once the cultured cells were harvested on day 6, the expression of the surface molecules CD11c, CD14, CD83, CD86, CD115, CD209 and HLA-DR was determined by flow cytometry. Cells were incubated for 20 min at room temperature in dark with the proper amount of the corresponding monoclonal antibodies (anti-): CD11c phycoerythrin (PE)-Cyanine dye 7 (PE-Cy7), CD14 Violet 450 (V450), CD83 allophycocyanin (APC), CD86 fluorescein isothiocyanate (FITC), CD115 PE, CD209 APC and HLA-DR Violet 500 (V500) (all of them from BD Biosciences, except CD209 APC from BioLegend, San Diego, CA, USA). Samples were acquired on a FACSCanto II flow cytometer and analyzed using the FACSDiva software (BD Biosciences).