Aging Aging

Human T-Cell Clones 57buffer. Load this eluate onto a second column and isolate the CD34 + cells isolatedby repeating the above procedure.4. Incubate the microbead-labeled CD34 + cell population with Multisort ReleaseReagent (Miltenyi Biotec) for 10 min at 12°C to release beads from the cells.5. Then load the suspension onto a third MS column in a magnetic field. Centrifugethe eluate containing bead-free CD34 + cells through a cushion of 10% BSA inPBS for 10 min at 600g. Resuspend pellet by adding 30 µL of stop reagent and10 µL of streptavidin-conjugated microbeads (Miltenyi Biotec) and then incubateat 6°C for 30 min.6. Resuspend cells in 500 µL of buffer and load onto freshly prepared MS columnsin a magnetic field. Collect eluate containing the CD34 + cells and check purity.For this, incubate the cells for 30 min at 4°C with phycoerythrin (PE)-conjugatedCD34 MAb directed against an epitope other than that used for bead separation.Greater than 98% of the cell population must be CD34 + .3.2. LD Cloning, Propagation, and Longevity Assessment1. Resuspend cells for cloning in culture medium (see Note 2) and adjust the concentrationsso that 10 µL contain 45, 4.5, or 0.45 cells. Pipet 10 µL of the 0.45suspension to 60 × 1 mm diameter wells of culture trays (“Terasaki plates”) andleave in a vibration-free area for 1 h. Check distribution of cells in the wellsvisually using an inverted microscope (being careful to look around the edges ofthe wells). Only 37% of the wells should contain cells according to the Poissondistribution. Readjust dilutions if necessary, replate, and check again.2. Plate multiple trays (at least five) with the 0.45 cells/10 µL suspension, one with4.5 and one with 45. Add feeder cells to each well. Irradiated (30 Gy) pooledPBMCs can most flexibly be used as feeder cells at 1 × 10 4 /well (see Note 3).3. Stack plates wrapped in aluminum foil for ease of handling and as a precautionagainst contamination. Incubate at 37°C in 5% CO 2 in air in a humidified incubatorfor up to a week and then examine the plates using an inverted microscope.4. Transfer contents of wells containing viable growing cells (> one third full) to7 mm diameter flat-bottom microtiter plate wells with fresh medium and 1 × 10 5feeder cells. Retain Terasaki plates for up to 2–3 wk and examine again at intervalsto identify any late positive wells. Transfer these to microtiter platesas well.5. Examine microtiter plates every few days. Split those becoming overcrowdedwith growing cells 1:1 into new culture wells and re-feed with medium (but notfeeder cells). After about a week in microtiter plates, transfer contents of wellswith growing cells into 16 mm diameter cluster plate wells with 2–5 × 10 5 feedercells, and fresh medium (see Note 4).6. Observe after 3–4 d. Divide wells that are full or nearly full into four, the othersinto two, with fresh media, but no more feeder cells. After a total of about a week incluster plates, count the number of cells in each clone and subculture to 2 × 10 5 /well,again with 2–5 × 10 5 feeders/well and fresh medium. Supplement with freshmedium after 3–4 d and subculture again if necessary. Continue to propagate by