Aging Aging

148 Clingen, Lowe, and Green3. Methods3.1. Preparation of Cellular MaterialResults in the Comet assay are critically dependent on gentle handling of thetest material. We have obtained acceptable results with the procedures outlinedin the following list. Where possible all procedures should be performed undersubdued lighting. Cultured cells are maintained at 37°C in a humidified atmospherecontaining 5% CO 2 . Typically 1–2 × 10 4 cells are required per slidewhen using 22 × 22 mm coverslips. Standard experiments of 16–18 slidesrequire a total of approx 2.5–5 × 10 5 cells.1. Whole blood samples may be obtained using a Soft Touch (Boehringer, Lewes,UK) or similar finger-pricking device and taken up using a Gilson-type disposabletip pipet (see Note 4). Blood (40 µL) is suspended in an Eppendorf tube with160 µL of clear RPMI 1640 containing four heparin-coated beads (taken from thebarrel of a blood sampling syringe). This yields sufficient cells for eight slideswhen diluted with 200 µL of 1.4% LMP agarose. Samples are held at room temperatureand slides made as quickly as possible.2. Mononuclear cell fractions (MNCs) from individual donors can be isolated fromlarger blood samples (5–50 mL) (see Notes 5 and 6). Blood is diluted 1:1 withRPMI 1640 and 10–15 mL carefully layered onto 10 mL Histopaque in centrifugetubes. These are centrifuged at 700g for 20 min at room temperature. TheMNCs are removed from the Histopaque/plasma interface with a sterile plasticPasteur pipet and approx 5 mL transferred into individual centrifuge tubes. Theseare made up to 20 mL with RPMI 1640 and centrifuged for 10 min at 700g. Thepellets are resuspended with 25 mL of RPMI 1640 containing 10% human ABserum and centrifuged for 10 min at 700g. The pellets are resuspended with 25 mLof RPMI containing 10% human AB serum and the number of cells counted. Thecells are centrifuged for 10 min at 700g, resuspended at approx 3 × 10 6 cells/mLin 10% DMSO/90% FCS and cryopreserved in 1-mL aliquots. Typically, 1 mL ofblood should yield approx 1 × 10 6 mononuclear cells. Prior to use for DNAdamage or repair experiments, cryopreserved mononuclear cells should be rapidlythawed, washed with 5 mL of complete RPMI culture medium, centrifuged at250g for 5 min, resuspended at 10 6 cells/mL in complete RPMI culture medium,and incubated at 37°C overnight (see Note 7).3. Primary human fibroblasts may be obtained from small skin biopsies and maintainedin tissue culture flasks in MEM culture medium (28). Cells are washedwith PBS and trypsinized with fresh trypsin-EDTA. It is essential to trypsinizefor a minimum period (typically less than 5 min at 37°C is sufficient). Twovolumes of MEM culture medium are added to inactivate the trypsin, and cellsare centrifuged at 250g for 5 min and resuspended in PBS at approx 10 6 cells/mL.For repair kinetic experiments on fibroblasts lasting more than 8 h it is necessaryto preincubate cells for at least 72 h in MEM containing 0.5% FCS to inhibit celldivision (see Note 8). Typically, 3 × 10 5 cells per dish can be maintained in anumber of 5 cm diameter tissue culture grade Petri dishes.