Aging Aging

Mutation and the Aging Process 1853. To score the plates, place each plate on the stage of an inverted microscope andcommence scoring from top left hand well down the remaining wells. Moveacross and score upwards. Exclude all wells in which evaporation has taken place.4. A well should be scored positive if more than 50 viable lymphocytes are present(see Note 6).3.7. Calculation of MF/10 6 cellsUsing the figures obtained (number of positive or number of negative wellsand total number of wells counted per plate) from the scoring of the CE platesin selective and nonselective conditions the following equations are used tocalculate MF/10 6 cells.CE in nonselective plates or selective plates = –ln (P 0 ) CE plate / no. of cells per wellWhere:Then,P 0 = no. of negative wells / total no. of wellsMF/10 6 cells = CE in selective plates / CE in nonselective plates4. Notes1. For each new batch of IL-2 purchased it is necessary to establish the optimalamount of IL-2 to support lymphocyte culture. To do this, a number of cloningefficiency plates (non-selective conditions) are set up using a range ofIL-2 concentrations, for example, 50, 100, 150, 200, 250, 300, 350, 400, and450 U/mL. The concentration at which optimum growth conditions (highestcloning efficiencies in nonselective plates) are achieved is then chosen foruse in all subsequent cloning assays until that batch of IL-2 runs out.2. Following collection, blood tubes, if they require transportation, should beprotected from sudden temperature changes and vibration, as these factorscontribute to low lymphocyte cloning efficiencies.3. An aspiration pump creates a vacuum to remove all unwanted layers fromthe separation process. A vacuum pump is attached to a Buchner flask, creatinga vacuum that extracts all unwanted materials through a glass Pasteurpipet.4. The same reagent reservoir can be used per sample for nonselective and selectiveplates provided that the nonselective plates are plated first, so as not be to contaminatedwith 6-TG.5. To reduce evaporation from the 96-well plates, place the plates inside a plasticstorage container with small holes to permit gaseous exchange.6. Viable cells are pleomorphic with regular borders and bright, nongranularcenters. They form tightly packed, regular shaped clumps. Dead feeder cellsand lymphocytes (various sizes with irregular borders and dark granular centres)also form clumps but these clumps are loosely packed.