Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

Begin with a chemically fixed tumor cell suspension.

Add an optimum concentration of tumor-binding immunoglobulin G that binds to tumor cell surface antigens, forming immune complexes, or ICs.

Transfer the ICs to a culture plate containing adhered dendritic cells. Incubate.

The dendritic cell recognizes the IC, internalizes it, and processes it into peptide fragments.

The fragments are loaded onto major histocompatibility complex class II or MHC-II molecules and presented on the cell surface, along with the costimulatory molecule CD86.

Remove media. Add a cell dissociation buffer and pipette vigorously to detach the cells.

Transfer the cells to a tube.

Centrifuge and resuspend the cells in a blocking solution to block non-specific interactions.

Overlay with a fluorophore-labeled antibody cocktail targeting MHC-II and CD86.

Add a DNA-binding dye and incubate briefly to stain dead cell nuclei.

Using flow cytometry, identify the live cells co-expressing MHC-II and CD86, confirming IC-mediated dendritic cell activation.