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Best Game PUBG[Flow cytometric method for monitoring the progress of natural killer cell-mediated cytotoxicity during long-term culture of human peripheral blood lymphocytes].
A method for monitoring the in vitro natural killer cell-mediated cytotoxicity using flow cytometry has been developed in this study. Peripheral blood lymphocytes from HLA-unrestricted donor were cultured in suspension with the interferon-containing IFN-tau (transforming growth factor beta) at 28 degrees C in the culture medium containing 15% AB serum. The peripheral blood lymphocytes were separated from their adherent cells after a 14-day culture and were activated by incubating with the culture supernatant of the EBV-transformed human ras-transfected Burkitt lymphoma cells containing 50-60% of natural killer (NK) cell activity. After the activation, the lymphocytes were stained with anti-CD16 monoclonal antibody and then evaluated with a FACS Vantage. The NK activity of lymphocytes was expressed as a percentage of the number of NK-sensitive K562 cells out of the total number of the T lymphocytes. The cultures which were started with different numbers of peripheral blood lymphocytes were set in order to establish a dose-response curve for the donor-dependent NK activity against K562 cells. The culture started with 5 X 10(5) cells/ml yielded the highest NK activity, when it was 2.5 X 10(5) cells/ml, when the NK activity decreased to 1 X 10(5) cells/ml, it no longer presented an increase. In addition, the culture was performed for a long-term culture for 14 days to observe the changes in the NK activity during the culture. The culture with 5 X 10(5) cells/ml yielded the highest NK activity during the 14-day culture. In addition, the culture was also performed using recipient-lymphocytes, which were separated from the donor-lymphocytes by the recipient anti-lymphocyte serum (R-ALS). The recipient-lymphocytes were activated by their culture with 500 000 irradiated killer-lymphocytes. Both the donor- and the recipient-lymphocytes demonstrated a dose-response curve to the change in the NK activity during the culture. This method can be used for monitoring the progress of NK activity of the
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