Aging Aging

Mutation and the Aging Process 183brake off on centrifuge). Following centrifugation gently aspirate (see Note 3) theupper layer using a sterile glass Pasteur pipet, to within 0.5 cm of the opaqueinterface containing the mononuclear cells (MNCs). Discard the aspirate.3. Gently aspirate the opaque layer containing the MNCs and transfer to a sterileuniversal containing 15 mL of RPMI 1640 (Gibco-BRL). Mix MNC suspensiongently by aspiration and then centrifuge at 250g for 10 min. Remove supernatantby aspiration and discard.4. Pool each MNC pellet (for each subject blood sample) and wash 1× with RPMI1640 (Gibco-BRL) at room temperature. Remove an aliquot, add trypan blue,and count viable cells using a hemocytometer. Recovery is generally in the regionof 2.5 million MNC/mL of whole blood.5. Resuspend MNCs in LCM at a concentration of 1 × 10 6 cells/mL and incubate overnightat 37°C, in a 5% CO 2 /air, humidified atmosphere. Monocytes will adhere to thesurface of the flask while lymphocytes remain in suspension in the culture medium.3.2. Cryopreservation of MNCsIt may not always be possible to perform the cloning assay on the same dayas the blood is collected. In such circumstances the MNC fraction can becryopreserved until required.1. Prepare freeze down medium (FDM): 90% FCS (not heat inactivated) and 10%glycerol.2. When the MNC fractions from one blood sample have been prepared (Subheading3.2., step 4) add FDM to achieve a final concentration of 3 × 10 6 MNCs/mL.3. Transfer the resultant cell suspension into 3-mL cryotubes and then place thetubes into a polystyrene box. Put the box in a freezer at –70°C overnight, thentransfer to liquid nitrogen for long-term storage.3.3. Thawing of Previously Cryopreserved LymphocytesIf cryopreserved, lymphocytes have to be gently thawed 24 h prior to cloning.1. Thaw the required number of vials rapidly in a 37°C water bath.2. Transfer the contents of the vials to a universal and dilute dropwise, slowly withcold RPMI 1640 (Dutch Modification) to a volume of 20 mL. Agitate the universalcontainer during addition of the RPMI.3. Centrifuge at 440g for 10 min. Remove supernatant and resuspend in warm RPMI1640 containing 5% FCS (heat-inactivated).4. Centrifuge at 440g for 10 min.5. Discard supernatant and resuspend pellet in warm culture medium at a concentrationof 1 × 10 6 cells/mL.6. Incubate overnight at 37°C in a 5% CO 2 /air, humidified atmosphere. After overnightincubation count lymphocytes using a hemocytometer. The number of lymphocytesper milliliter is usually less than 1 × 10 6 . A successful thaw will yield 7–9 × 10 5 cells/mL.A count of less than 4 × 10 5 cells/mL should not be used for cloning.