After recovering CD8 cross-primed from coculture, centrifuge them at 1100 g for 10 minutes in 10 milliliters of complete growth medium. Resuspend 1 x 10 to the power of 7 total cells in 100 microliters of dead cell removal microbeads, and incubate for 15 minutes at room temperature. Place separation columns in the magnetic separator, and rinse them with binding buffer.
Transfer cell suspensions into separation columns, and collect the flow-through. After washing the column, collect the unlabeled cells that pass through, and combine them with previously collected flow-through. Centrifuge enriched live CD8 cross-primed at 1100 g for five minutes in a complete growth medium.
After counting, culture CD8 cross-primed cells with fresh MCA205 cells at a two-to-one ratio in 12 well plates, and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Before recovering CD8 effector, add monensin and brefeldin to the cancer immune cell cocultures for six hours. Then recover CD8 effector, and centrifuge twice at 1100 g for five minutes.
Resuspend cells in three different primary antibody mixes prepared in cold FACS buffer, and incubate for 20 minutes at four degrees Celsius, protecting from light. Add 100 microliters of fixation solution to the cell palate from Mix C and incubate for 20 minutes at four degrees Celsius in the dark. Resuspend the cells in a cold wash buffer, and incubate for 30 minutes at four degrees Celsius.
After washing cells twice in FACS buffer, add 100 microliters of PBS containing the Vitality Fixable Aqua Dye to cell pellets for 30 minutes. Identify 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 525/50 nanometer emission band-pass filter and SSCA to remove dead cells, Using 660/20 and 780/60 nanometer emission band-pass filters, detect cell positivity for CDAA and CD3 in two parameter density plots. Further, analyze cells for CD44, CD25, and CD69 markers for CD137 marker, and for interferon gamma positive and granzyme B for Mix A, B, and C, respectively. For tumor killing assay, supplement the coculture medium with a real-time cell death quantification dye.
After warming the plate to 37 degrees Celsius in the live cell analysis system, perform data scanning every two hours for up to 72 hours. Centrifuge the cells twice at 1000 g for five minutes. Stain cells for surface markers with 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees celsius in the dark.
Then add the vitality dye propidium iodide to stain the cells for a gating strategy. 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.
Use a 450/50 nanometer emission band pass filter to detect cancer cells negative for CD45. In a single parameter histogram, using a 610/620 nanometer band-pass emission filter, analyze cells for propidium iodide incorporation as mean fluorescent intensity. Through multi-parameter flow cytometry, the surface expression levels of the early activation marker CD69, late activation markers CD44 and CD25, membrane expression of CD137, and CD107, tumor reactivity markers were enhanced.
The intracellular levels of cytotoxic molecules, granzyme B, and interferon gamma positive were progressively and significantly increased, reaching a peak of expression at 72 hours of coculture. Cognate cocultured MCA205 cells underwent significant levels of CD8 effector mediated death.
