Aging Aging

T-Cell Function in the Aged 2873. Dispense the anti-CD2 MAbs and graded amounts of cofactor(s) to microtiterwells, add 2 × 10 5 T-cells per well, and culture for 3–4 d. Thus the capacity ofeach cofactor to induce activation is easily tested. Anti-CD28 MAbs are excellentcostimulatory reagents, presumably because they mimic the key CD28–CD80signal transduction pathway.3.4. RT/PCR AnalysisWe have established a reliable and semiquantitative RT-PCR technique thatis based on taking several 5-µL aliquots during the linear phase of the PCR andrelating the amount of target product to two control genes, actin and CD3 δ, fora given starting cell number. However, good commercial kits are now availablefor quantitative RT-PCR that are designed for a number of cytokines.1. After 4–48 h of culture, harvest as few as 2 × 10 5 and up to 1 × 10 6 stimulated andnonstimulated (control) T cells from 24-well Costar plates and wash twice inPBS-containing 0.01% diethylpyrocarbonate (DEPC) (an RNase inhibitor) (seeNote 11).2. To extract cytoplasmic RNA, pellet the cells in Eppendorf tubes and remove asmuch PBS as possible. Lyse the cell pellets in 0.1 mL of solution A (10 mMTris, pH 7.5; 150 mM NaCl; 0.65% NP-40; and 10 mM vanyl ribonucleosidecomplexes). After about 10 s, centrifuge the tubes at 12,000g for 1 min andtransfer the supernatant containing the cytoplasmic fraction (be careful not todisturb the pellet) to a new tube containing 0.3 mL of solution B (10 mM sodiumacetate, pH 5.0; 50 mM NaCl; 5 mM EDTA; and 0.5% sodium dodecyl sulfate[SDS]).3. Vortex-mix the mixture and then extract twice with 800 µL phenol/chloroform(1:1) and finally once more with chloroform alone.4. Remove about 40 µL of the aqueous phase and use an aliquot for the cDNA reaction.Store the remainder at –80°C as 1250 or 2500 cell equivalents per microliterof supernatant. The RNA is stable for at least 12 mo.5. First-strand cDNA is synthesized from 5 to 10 µL of RNA supernatant, usingM-MLV reverse transcriptase (BRL) and oligo dT priming according to themanufacturer’s directions, in a final volume of 40 µL. The reaction is carried outat 37°C for 1 h.6. One eighth of the cDNA is then added to 45 µL of PCR mix and the tubes subjectedto 27–34 cycles of PCR amplification using a thermal cycler with a 1 min/95°C denaturation, 2 min/60°C annealing, and 3 min/72°C extension profile.7. A 5 µL aliquot is taken at the end of several cycles (e.g., 27, 29, and 31), mixedwith loading buffer, and then analyzed by electrophoresis on 2% agarose gelsand subsequently stained with ethidium bromide (Et Br). A 123-basepair DNAladder (Gibco-BRL) is used as a marker. Compare and contrast each gene productwith the actin and CD3 δ product. Thus its possible to screen about 30–40different genes from a single cell preparation. For relevant primer sequencessee (1).