H22 cells were labeled with 10 μM DiD (Sigma-Aldrich, USA) after the induction of apoptosis and serial centrifugation. DiD-labeled intact cells or apoptotic body and blebs (50 μg/mL) were added to 1 × 106/mL DCs with a final volume of 500 μL and subsequently incubated at 4°C or 37°C for 2 hours. Next, DCs were collected and labeled with FITC conjugated anti-CD11c antibody (BioLegend, San Diego, CA). Internalization of intact cells or apoptotic body and blebs by DCs was visualized with confocal microscope. In addition, the percentage of FITC/DiD double-positive cells was analyzed by flow cytometry.
For the analysis of DC maturation, 5 × 105 immature DCs were incubated in medium alone or medium supplemented with intact cells or apoptotic body and blebs. Addition of 1 μg/mL lipopolysaccharide (LPS) (Sigma-Aldrich, USA) was used as a positive control for the maturation of DCs. After 16 hours of incubation, cells were harvested and stained with FITC conjugated anti-CD11c antibody and PE conjugated anti-CD86 antibody (BioLegend, San Diego, CA). Cells were subjected to flow cytometry and acquired data were analyzed by FlowJo software.
For the analysis of DC maturation, 5 × 105 immature DCs were incubated in medium alone or medium supplemented with intact cells or apoptotic body and blebs. Addition of 1 μg/mL lipopolysaccharide (LPS) (Sigma-Aldrich, USA) was used as a positive control for the maturation of DCs. After 16 hours of incubation, cells were harvested and stained with FITC conjugated anti-CD11c antibody and PE conjugated anti-CD86 antibody (BioLegend, San Diego, CA). Cells were subjected to flow cytometry and acquired data were analyzed by FlowJo software.