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.

First, fix the cells in 1.8% buffered paraformaldehyde for 10 minutes at room temperature. Seed the cell suspension in each well of a U-shaped 96-well plate. Next, add varying dilutions of tumor-binding antibodies in each well. Thereafter, incubate the cells on ice for 15 to 20 minutes.

After incubation, wash the cells with 150 microliters of phosphate-buffered saline, then centrifuge at 400 times g for 5 to 10 minutes at 4 degrees Celsius. Post-centrifugation, expel the supernatant and wash the cells twice. Dissolve the cells in 100 microliters of FACS buffer, containing fluorophore-conjugated secondary antibody. Next, incubate the plate on ice for 15 to 20 minutes.

After 15 to 20 minutes, add 200 microliters of FACS buffer to wash the cells. Centrifuge the plate at 400 times g for 5 to 10 minutes. Decant the supernatant. Conduct flow cytometry to analyze the tumor binding, and to determine the minimal concentration of immunoglobulin G required to coat the cells.

Once the cells are coated with minimal immunoglobulin G concentration, add the CFSE-labeled tumor immune complex to the monocyte-derived dendritic cells in a 1:5 ratio. Then, incubate the mixture for 12 to 16 hours overnight in complete medium. Perform FACS analysis of the monocyte-derived dendritic cell activation.