Aging Aging

NK Cell Function in Aging 3173.3. Cytotoxic Assays for NK Cells: Preparation of Target CellsSeveral cell lines are used as target cells for measuring NK lysis. The mostcommonly used are K562 cells, as well as C1R or 721.221. Daudi cell line, anNK-resistant cell line, is used to study cytotoxicity by IL-2-activated NK cells.Furthermore, P815, an NK-resistant mastocytoma murine cell line whichexpress high concentrations of Fc receptors, can be used to analyze redirectedlysis, for example, lysis of the target in the presence of antibodies against NKtriggering structures. Both Daudi and P815 cell lines can be used to analyzeADCC by using NK cells and IgG antibodies specific for the target cells. Growthese cell lines in RPMI 1640 + 10% FCS, 2 mM glutamine, 100 U/mL ofpenicillin, and 10 µg/mL of streptomycin and use them during the logarithmicgrowth phase (Note 5).1. Radioactive labeling of target cells with 51 sodium chromate: Use 51 Cr solution innormal saline to label target cells and no later than 15 d after the reference day.For target cell labeling, wash 1–2 × 10 6 once with fresh medium, remove thesupernatant, and incubate pellet with 100 µCi 51 Cr in differing volumes, accordingto the decay table (Note 6). Incubate the cells at 37°C in 5% CO 2 for 1 h withoccasional shaking at 10–15-min intervals. During following procedures, keepthe target cells on ice to reduce spontaneous isotope release. Wash the target tumorcells 3× at 4°C in cold RPMI 1640 + 10% FCS and resuspend in medium at aconcentration of 5 × 10 4 cells/mL (Note 7).2. NK cell cytotoxicity assays: The effector/target cell ratio will depend on thenature of the effector population and its level of cytotoxic activity. A startingeffector/target ratio of 100:1 or 50:1 will be required for assaying freshly isolatedNK cells, whereas for purified NK cells or activated LAK this may be at most20:1 or 10:1. In addition to NK and LAK assays ADCC can be determined whenan NK-resistant cell line is used and anti-target antibodies are added to the assay.Redirected lysis is measured when P815 are used as targets and MAbs againstNK triggering structures (i.e., CD16, CD69) are added to the assay. Perform thecytotoxicity assays in V-bottom 96-well microtiter plates with a final volume of200 µL. Seed each effector/target cell ratio (E/T) in triplicate. For spontaneousrelease control, samples of target cells are resuspended in medium alone. Maximumor total release of 51 Cr from the target cells is obtained by mixing 5 × 10 3labeled target cells with 100 µL of 2% Triton X-100. Use at least six replicatewells to evaluate spontaneous and maximum release. Seed serial dilutions ofmononuclear effector in 100 µL of culture medium and 100 µL of 51 Cr-labeledtumor target cells with an E/T ranging from 100:1 to 12:1 (Note 8). Centrifugethe plates at 4°C for 3 min and incubate at 37°C in 5% CO 2 for 4 h. Harvest andtransfer to a tube 100 µL of culture supernatant at the end of the incubation time.Determine 51 Cr release in a γ-counter. Calculate specific 51 Cr release accordingto the formula: