Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer
Transcrição
Suspend mouse or human primary NK cells in the wells of a 24-well plate at 0.5 x 105 per milliliter of medium with GFP lentivirus supernatant at an MOY of 5, 10, and 20 and in the presence of polybrene, protamine sulfate, or dextran.
Centrifuge the plates at 1,000 times g for 60 minutes. Then, without decanting the supernatant, culture the cells in a 37 degrees Celsius incubator infused with 5.2% carbon dioxide overnight. After the cells have been washed with 10 milliliters of PBS, use 2 milliliters of RPMI 1640 medium with IL-2 to resuspend the cells.
The day before harvesting the cells, coat 96-well highly protein absorbent polystyrene plates with 2.5 micrograms per milliliter of anti-NKG2D monoclonal antibodies, and incubate the plates at room temperature or in the fridge overnight. The following day, use 100 microliters of PBS to wash each well three times.
Harvest the transduced NK cells by first gently tapping the plates. Then add 100 microliters of the cells to each well of the 96-well plates. 16 to 18 hours post-activation, use a multichannel pipette to collect the supernatants. Then, using the recombinant cytokines from an ELISA kit, generate a standard curve and quantify the cytokines such as interferon-gamma in the supernatant.
To test the NK cell viability, four days after transduction, wash the cells with 10 milliliters of cold PBS two times. Then, harvest the cells and use an annexin-V/7-amino-actinomycin D to stain them. Use flow cytometry to determine the percent of necrotic cells among the transformed NK cells, and analyze the data according to the text protocol.
