Transwell Migration Assay and Analyses of Activation of DCs

At day 10 of DC cultivation, DCs were harvested, counted, and adjusted to 1.25 × 10⁶ DCs/ml DC medium. SN from the irradiated CT26 cells were thawed on ice and, afterward, 1.5 ml SN per approach was placed in the bottom of a well of a six-well plate (Greiner Bio-One, Frickenhausen, Germany). A cell permeable membrane (with 3.0-μm pore size; Greiner Bio-One, Frickenhausen, Germany) was attached to each well and 800-μl DC cell suspension (containing 1 × 10⁶ cells) was transferred on the upper side of the membrane. The six-well plates were stored in a cell incubator at 37°C overnight (14 h).

For analysis by flow cytometry, migrated cells had to be collected. Therefore, each membrane was carefully lifted with tweezers, and the bottom side was washed with cell suspension of the respective well to collect these cells. Then, the cell suspension was collected from each well and strongly adherent cells were removed by rinsing the well with cold PBS. After centrifugation, each cell pellet was resuspended in Fc block solution [PBS, 10% inactivated FBS, 0.001% Fc-Block, CD16/32 (ebioscience, Frankfurt, Germany)] and incubated for 10 min at 4°C in the dark to prevent non-specific binding of antibodies to Fc receptors.

Cell suspension was distributed to three 1.4 ml PP tubes (Micronic, AR Lelystad, The Netherlands) and antibody solution [MHCII-e450 (0.4 μg/ml, eBioscience, Frankfurt, Germany), CD80-PE (0.4 μg/ml, BD Pharmingen, New York, NY, USA), and CD86-Alexa^® Fluor700 (0.4 μg/ml, BD Pharmingen. New York, NY, USA) diluted in FACS buffer (PBS, 2% inactivated FBS)] was added. After incubation for 30 min at 4°C in the dark, cells were washed with FACS buffer and resuspended in it. Further, SN were also directly added to DCs and the expression of the activation markers CD80 and CD86 was analyzed similarly 24 and 48 h afterward. Cells were analyzed by flow cytometry (Gallios, BeckmanCoulter Inc., Krefeld, Germany), and the number of MHCII⁺ cells was defined as the number of migrated DCs. Gating on MHCII⁺ cells was performed for analysis of the mean fluorescence intensity of cells stained with maturation markers CD80 and CD86.