Aging Aging

316 Mariani, Alonso, and Solanasubpopulations of white blood cells (Note 3). Optimal gating for the identificationof lymphocyte populations requires polygonal computer-generated windows(Note 4). FITC green fluorescence or PE red fluorescence can be simultaneouslyanalyzed. For each sample, 10 4 gated cells are usually analyzed.3.2. Cytotoxic Assays for NK Cells: Preparation of Effector CellsDifferent sources of effector cells can be used as effectors of NK cell cytotoxicity.In age-related studies plastic- and nylon-wool-depleted PBMCs arecommonly used to analyze NK killing of K562 target cells whereas IL-2-activatedcells are used to study LAK killing of the NK-resistant Daudi cell line.1. PBMC enriched by depletion of plastic and NW adherent cells. Incubate PBMCsat 5 × 10 6 PBMCs/mL in a Petri dish for 60–90 min at 37°C in 5% CO 2 , formonocyte depletion. Recover nonadherent cells, wash twice with PBS, and dilutein 2 mL of medium. The cells are then incubated in prewashed nylon wool for45–60 min at 37°C in 5% CO 2 , for B cell depletion. Recover the nonadherent(T/NK cells) by washing the column with 20 mL of culture medium and resuspend.This cell population contains 20–30% of CD56 + NK cells.2. Activation of NK cells with IL-2. Culture the cells obtained as indicated instep 1 in 5% CO 2 at 37°C for 7–10 d in the presence of 500 U/mL of IL-2,without further activation requirements. This population is considered LAKcells and is constituted by a mixture of activated NK and T-cells able to lyseNK-resistant cell lines.3. Purification of NK cells by immunomagnetic separation. Incubate the nonadherentcells (T/NK cells) obtained as indicated in step 1 with anti-CD3 MAb for 30 minat 4°C and add 0.7–0.9 × 10 8 of GAM-coupled magnetic beads. Incubate for 30 minat 4°C under gentle shaking for T-cell depletion. Remove the cells rosetting withthe beads by using a magnetic particle concentrator (MPC1) and collect the supernatantwith the CD3-negative cells. Alternatively, whole PBMCs (5 × 10 6 /mL) canbe directly incubated with CD3, CD4, CD19, and CD14 MAb for 30 min at 4°C,and then with GAM-beads for 45 min at 4°C. Cells and beads should be gentlymixed throughout. After the incubation with beads, use a magnet to separate beadswith attached CD3/CD4 T-cells, CD14 monocytes, and CD19 B cells from NKcells. Centrifuge and wash with complete medium the recovered free cells. Thiscell population should be 75–95% CD56 + and CD16 + ,