Aging Aging

162 van der Schans10. High-binding polystyrene microtiter plates, Costar, 96-well, cat. no. 3590/9018.11. Pipet tips (white opaque), 1–200 µL, Costar, no. 4862.12. Pipet tips (blue), 100–1000 µL, Eppendorf, no. 0030 015.002.13. Pipets, P100, P200, and P1000, Gilson.14. 12-Channel pipet, 25–200 µL; Micronic (Macap) cat. no. 200–12.15. 96-Well dispenser, Transtar 96, adjustable volume, cat. no. 7605 and Transtarelevator cat. no. 7606, Costar.16. Plate washer, Skanwasher 300, Skatron.17. 12-Channel handplate washer, with tube connected to a polypropylene flaskwith PT.18. Plate vibrators, Titertek (Flow) and IKA (model MTS4).2.4. Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)1. 1 M NaOH solution: Dissolve 3.93 g of NaOH (in tablet form) in (by weighing)98 g MQ-water (or proportionally other amounts) in a 100-mL medium flask.Close with two tissues of parafilm covered with a loosely tightened screwcap.Can be stored at room temperature for 2 mo.2. 1.3 M NaCl solution: Dissolve 38.0 g of NaCl in MQ-water and fill up to 500 mL.Can be stored at room temperature for 2 mo.3. Alkali-solution: Add 2.00 mL of 1 M NaOH to 103 g of a 1.3 M NaCl solution(calculated pH 12.3).4. Tween-20 (polyoxyethylenesorbitan): Sigma cat. no. P-1379.5. 10× PBS solution: 81.8 g of NaCl, 14.4 g of Na 2 HPO 4 .2H 2 O, 2.0 g of KH 2 PO 4 ,and 1.9 g of KCl in 1 L of MQ-water. Check the pH, which should be between 7.0and 7.4. Can be stored at room temperature for 2 mo.6. PBS solution: Add to a 10-L polyethylene vial, with valve, 9 L of MQ-water and1 L of 10× PBS, and mix thoroughly.7. PT: Add to a 25-L polyethylene flask, with valve, 18 L of MQ-water, 2 L of 10×PBS, and 10 g of Tween-20, and mix thoroughly.8. FCS: Gibco-BRL, cat. no. 10106–110, stored at –20°C in 500-mL flasks.9. hiFCS: Thaw a 500-mL flask with FCS in a 37°C water bath. Divide under sterileconditions (in a laminar flow cabinet) in 30-mL flasks and/or 50 10-mL polystyrenetubes with cap. Incubate all flasks and tubes for 0.5 h in a 56°C water bath.Place in refrigerator. Can be stored for 2 mo (2–8°C).10. Sodium dodecyl sulfate (SDS): Sigma, cat. no. L-4509.11. 10% SDS solution: Dissolve 10 g of SDS in 100 mL of MQ-water. Can be storedat room temperature for 2 mo.12. PT + 0.05% SDS + 5% hiFCS: Make up solution of PT, hiFCS, and 10% SDS inthe ratio of 1 mL of hiFCS, 19 mL of PT, and 100 µL of 10% SDS. Prepare thissolution no sooner than 1 h before use.13. D1B-AP conjugate: Dilute D1B-AP conjugate, prepared as described elsewhere(2), to the optimal prescribed dilution (1000–40,000×; see Notes 4 and 10) in PT+ 0.05% SDS + 5% hiFCS. Prepare this solution no sooner than 1 h before use.