Aging Aging

T-Cell Function in the Aged 285a range of concentrations, to triplicate wells of a 96- or 24-well plate, respectively(see Note 3).2. Alternatively or in parallel experiments, add titrated amounts of SEB (a finalconcentration of 10 ng/mL is optimal in my laboratory) or MAbs T112 andT113 for anti-CD2 stimulation (as a guide we use a final dilution each of 1:500ascites fluid).3. Dispense 180 µL or 900 µL of PBL (prewashed twice in PBS and resuspended inCCM at 5.5 × 10 5 /mL) to each 96- or 24-well plate, respectively, and incubate at37°C in a 5% CO 2 controlled atmosphere for 3–4 d. Try a range of cell concentrationsranging from 2 × 10 5 /mL to 1 × 10 6 /mL (see Note 4).4. At the designated time(s), appropiate cultures are spiked with 0.5 µCi [ 3 H]TdRfor 4 h before harvesting the cells using a semiautomated sample harvester. Theamount of [ 3 H]TdR incorporation is measured in a beta scintillation counter (subtractthe cpm recorded in negative control wells, i.e., wells containing nonactivatedcells).Cells are also harvested from parallel cultures at identical times for other analyses,for example, RT-PCR, cell cycle, and/or immunohistochemical analysis (seeNote 5). Remember to store the centrifuged culture supernatants at –80°C forcytokine ELISAs, if required.Alternatively, T-cell responses generated in one-way mixed lymphocyte reactions(MLR) using PBLs are analyzed by coculturing 1 × 10 5 cells (respondercells) from one individual with an equal number of irradiated (2000 rads) or mitomycinC-treated cells (stimulator cells) from one or more unrelated individualsin triplicate round-bottom 96-well plates in a final volume of 200 µL (see Note 6).Incubate for 7 d and spike with 0.5 µCi [ 3 H]TdR per well for 16–18 h beforeharvesting as described previously.3.2. T cell Activation Using Purified T Cells and AC1. Cultures are basically set up as described previously except purified T-cells areemployed (see Note 7); however, in these experiments the proportion of ACs ormonocytes to T-cells is controlled for each subject.2. We usually add 10% irradiated (2000 rads) AC-enriched cells (i.e., nylon wooladherentand plastic-adherent peripheral blood mononuclear cells) to each wellcontaining 1 × 10 5 or 1 × 10 6 T-cells (see Note 8).Again, it is often informative to try a range of AC concentrations (i.e., 5%to 30%).Furthermore, these experiments provide a degree of flexibility. That is, theACs can be either (1) irradiated autologous ACs (2) irradiated heterologousACs (a MLR is avoided here by culturing for 3 d) (3) transformed cell linessuch as K562 or U937 (irradiated with 4000 rads to prevent outgrowth) (4)irradiated CHO transfectants expressing specific human ligands singularly orin combination, for example, HLA DR, CD80, CD86, LFA-3, CD40, Fas, etc.,or (5), irradiated OKT3–hybridoma cells. Interestingly, the OKT3 expressing