Aging Aging

92 Kirk and Millersufficiently diluted there will be incomplete cell pelleting. Dilute further inHBSS–BSA if supernatant shows any turbidity following centrifugation.8. Resuspend lymphocytes in 15 mL of ice-cold HBSS–BSA and add to anti-Igcoateddishes. Incubate at 4°C on a level surface for 40 min. Swirl plates once atthe end of the first 20 min to redistribute the cells. Dishes of 150 mm diametershould receive approx 120–150 × 10 6 erythrocyte-depleted spleen cells; using morecells per dish diminishes cell purity, while using fewer cells diminishes cell yield.9. Gently separate nonadherent from adherent cells by swirling the plate vigorouslyand then slowly removing the cell suspension from the tilted dish. It is particularlyhelpful to remove the last 0.5–1 mL very slowly, allowing the meniscus tocollect at the low point of the dish. Wash plate once with 5 mL of ice-cold HBSS–BSA to improve cell yield, again taking care to remove the suspension slowly.Centrifuge cells at 4°C for 5 min at 500g.10. Resuspend cells in ice-cold HBSS–BSA and count. From 25 to 30 × 10 6 T-cellscan be obtained from a single spleen. Remove an aliquot of cells to perform flowcytometry with an antibody to mouse CD3ε to determine the proportion of T-cellsin the resulting population (typically 85–90%).3.1.2. Negative selection of T cell subsets1. Dilute T-cells, prepared as described in Subheading 3.1.1., into HBSS–BSA to aconcentration of approx 5 × 10 6 cells/mL.2. Add antibodies to cell markers of subsets to be depleted (i.e., for CD4 + T-cellpurification add antibodies to CD8). The amount of antibody needed is based ontrial experiments in which CD8 cell contamination of resulting cells is determinedby flow cytometric analysis. In our experiments, we use 1 µg of antibodyfor 10 7 cells at a concentration of 5 × 10 6 cells/mL.3. Incubate on ice for 20 min with gentle shaking at 5- to 10-min intervals.4. Prepare anti-rat IgG-coated beads according to the manufacturer’s specifications.Prepare enough for a bead-to-cell ratio of 50:1. Resuspend beads in 0.4 mL ofice-cold HBSS–BSA.5. Centrifuge cells at 4°C for 5 min at 500g. Wash cells once in 10 mL of coldHBSS–BSA.6. Resuspend cells in 1 mL of cold HBSS–BSA and add 0.4 mL of bead solution.7. Incubate on ice for 20 min with shaking every 5 min to ensure suspension ofbeads.8. Fill tube to 5 mL with cold HBSS–BSA and pass under magnetic field (PerSeptiveDiagnostics, Cambridge, MA, USA), which is kept at 4°C.9. Carefully remove cell suspension without disturbing the beads aligned along theside of tube.10. Repeat steps 8 and 9 two to three more times to ensure complete removal of beads.11. Centrifuge cells at 4°C for 5 min at 500g. Resuspend in cold RPMI supplementedwith 10% fetal calf serum (FCS) (RPMI–FCS) and count. An aliquot of ~0.5 × 10 6cells should be removed at this stage for flow cytometric analysis to assess thepurity of selected cells.