Aging Aging

56 Pawelec3. Method3.1. Source of Cells to Be Cloned3.1.1. Purification of T Cells (see Note 1)1. Selectively deplete non-T-cells from PBMC using a cocktail of antibodies forCD14 (expressed by monocytic cells, macrophages, dendritic cells), CD16 (onNK cells), CD19 (B cells and B-cell precursors), and HLA-DR (monocytes, Bcells, activated T-cells, and dendritic cells).2. Incubate 10 7 cells/mL at 4°C for 30 min with approx 10 µg/mL of each antibody,centrifuge, wash twice, and resuspend in 1.5 mL of phosphate-buffered saline(PBS) with 0.1% bovine serum albumin (BSA).3. Add approx 10 8 washed Dynabeads in 0.5 mL and incubate at room temperaturefor 1 h. Gently shake occasionally. Add 2 mL of PBS and put the tube into themagnetic field for 1–2 min. Gently aspirate the supernatant. This containsthe negatively selected cells not held by the magnet. Wash twice and controlpurity with anti-T-cell antibody by immunofluorescence.4. Remove any possible remaining functional accessory cells by treating the populationwith L-leucyl-L-leucine methyl esther (LME). Incubate at 2.5 × 10 6 /mL in10 mM LME for 45 min at room temperature in culture medium without serum.5. Wash twice and then check absence of functional accessory cells. This can bedone by stimulating with T-cell mitogens such as phytohemagglutinin in theabsence of added cells. There should be no response. After reconstitution of accessoryfunction with B-lymphoblastoid cell lines (B-LCLs) the response should bemeasurable. A typical protocol is to incubate 2.5 × 10 4 T-cells per round-bottommicrotiter plate well in culture medium together with 1% phytohemagglutinin(PHA, M form; Gibco-BRL) in triplicate. A duplicate set of wells receives inaddition 2.5 × 10 4 B-LCL cells (irradiated at 80 Gy). Proliferation can be assessed3 d later, for example, by addition of 37 kBq/well of tritiated thymidine andassessing incorporated nuclear radioactivity after 8–16 h.3.1.2. Purification of CD34 + Cells1. Separate low-density mononuclear cells (MNCs,