To begin, plate 3 times 10 to the power of 6MCA205 fibrosarcoma cells in 10 milliliters of complete growth medium in a 100 millimeters Petri dish. Irradiate cells with ultraviolet light and incubate at 37 degrees Celsius with 5%carbon dioxide for at least six hours to let them die. Wash in vitro differentiated dendritic cells, or DCs, twice, at 1100G for four minutes in a complete growth medium.
Count the bone marrow-derived empty DCs using the Trypan blue dye exclusion test. Centrifuge the irradiated cancer cells at 1100G for five minutes. Set the co-culture of empty DCs with irradiated apoptotic cells at a two to one ratio in a 12-well plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
The next day, wash the cells twice at 1100G for five minutes in 10 milliliters of complete growth medium in 15 milliliter tubes. For cell surface staining, re-suspend the bone marrow-derived DCs in 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees Celsius in the dark. After washing the cells with FACS buffer, add 100 microliters of PBS containing vitality fixable near-infrared dye for 30 minutes.
Wash the cells once with PBS before re-suspending them in the FACS buffer. Identify the cells of interest based on their FSCA and SSCA properties. Then plot FSCA and FSCH to exclude cell doublets and clumps from the analysis.
Plot 780 to 60 nanometer emission band pass filter area and SSCA to remove dead cells. Using 575 to 26 and 530 to 30 nanometer emission band pass filters, detect cell positivity for CD11C marker and PKH67 fluorescent cell linker in two parameter density plots. After counting, culture the CD8 T cells with bone marrow-derived DCs that had taken up apoptotic MCA205 cells at a two to five to one ratio at 37 degrees Celsius and 5%carbon dioxide for 72 hours.
Add EdU to the co-culture medium at 10 micromolar final concentration and incubate for 16 to 20 hours. Centrifuge the CD8 cross prime twice at 1100G for five minutes and stain for surface markers in cold FACS buffer for 20 minutes at four degrees Celsius in the dark. After washing the cells twice in FACS buffer, re-suspend them in 100 microliters of PBS containing the vitality fixable aqua dye for 30 minutes.
Wash the cell suspensions once with three milliliters of 1%BSA in PBS in flow tubes. Add 100 microliters of 4%paraformaldehyde for 15 minutes for cell fixation. Incubate the cells in 100 microliters of saponin-based permeabilization and wash reagent for 15 minutes.
Add 500 microliters of the reaction cocktail and incubate for 30 minutes in the dark. After washing, re-suspend the cells in 100 microliters of saponin-based permeabilization and wash reagent. Identify the cells of interest based on their FSEA and SSEA properties.
Then plot FSEA and FSEH to exclude cell doublets and clumps from the analysis. Plot 525 to 50 nanometer emission band pass filter area and SSCA to remove dead cells. Using 530 to 30 and 575 to 26 nanometer emission band pass filter density plots, detect cells'positivity for CD8a and CD3.
Analyze cells for Alexa Fluor 647 EdU incorporation in a single parameter histogram using a 660 to 620 nanometer band pass emission filter. CD11c positive dendritic cells effectively engulfed apoptotic MCA205 cancer cells at 37 degrees Celsius, demonstrating temperature-dependent phagocytosis in early cancer immunity cycle stages. After phagocytosis, dendritic cells showed increased levels of CD86 and MHC-II along with reduced levels of PDL1.
The clonal expansion of CD8 positive T cells once activated by mature dendritic cells was evident with approximately 20%proliferation rate. Using tumor-on-chip models, the chemotactic response of these T cells towards cancer cell released alarmins was observed.
