Aging Aging

288 BeckmanHaving identified a candidate gene(s) it is important, however, to examinenot just message but where possible, protein levels (ELISA) and functionalintegrity (bioassay).3.5. T-Cell Adherence to Endothelial Cells (ECs)This simple assay measures the capacity of resting and activated T-cells toadhere to activated or resting endothelium (see Note 12).3.5.1. Preparation of Human EC1. Human ECs are prepared by washing intact human umbilical veins, one endclosed with a sterile stopcock and valve, with 20 mL of Hanks’ balanced saltsolution (HBSS) containing 0.08% collagenase.2. After 10 min at 37°C, 20 mL of medium 199 is injected into the vein and thecollagenase solution washed out. The vein is massaged with fresh medium andthe stripped EC collected by centrifugation.3. ECs are cultured in medium 199 supplemented with 20% FCS and 20 µg/mLEndothelial Cell Growth Factor Supplement (Integrated Sciences) in flasks(Costar) precoated with 2.5 mL of gelatin (a 2% solution in HBSS).4. To remove the ECs from a flask, treat with 5 mL of trypsin/EDTA solution for5 min, then wash twice and resuspend the cells in 3 mL of the above medium.5. Add 1 mL per well to a 24-well culture plate containing a sterile round glasscoverslip (sterilized by soaking in ethanol, flaming, and washing in sterile PBS).After 2 d the cells form a new monolayer.6. Alternatively, monolayers can be derived from the spontaneously transformedimmortal human endothelial cell line, namely ECV304, by culturing cells at2 × 10 5 /mL in 24-well plates in medium 199 plus 10% FCS.7. To prepare activated EC monolayers, incubate with 5 ng/mL of tumor necrosisfactor-α (TNF-α) for 4 h and then wash the cells three times with PBS.3.5.2 Adherence Assay1. Dispense half a million resting or activated pan T cells (e.g., activation canachieved by incubating the cells with plate-bound MAb 64.1 for 4 h) to theEC-coated glass coverslips.2. After 90 min at 37°C wash the coverslips twice in PBS and incubate with 200 µLof MAb; for example, anti-CD3, anti-CD4 (helper T cells), anti-CD8 (supressor/cytotoxic T cells), anti-CD45RA (naive T cells), anti-CD45RO (memory T cells),or irrelevant MAbs (negative controls).3. Incubate for 20 min at 4°C and then wash the coverslips twice with PBS.4. Add 200 µL of pretitrated FITC-labeled goat anti-mouse IgG per well for 20 minat 4°C.5. Wash the coverslips thrice and remove them from the wells using a needle with abent tip and place on a glass slide for subsequent examination by UV microscopy.T-cells bound to the EC monolayers are easily identified by their stainingpattern with the appropriate MAb.