Aging Aging

Susceptibility of LDL to Oxidation 2011. Fasting peripheral venous blood is taken into 10-mL glass tubes containinglithium heparin (50 kU/L). Plasma is isolated by centrifugation (1500g for10 min) and stored in 1-mL aliquots at –70°C until required. These samples arestable for up to 6 mo (see Note 1).2. Polyallomer belltop centrifuge tubes (3 mL; 13 × 32 mm; Beckman cat. no.349621).3. Potassium bromide; 0.4451 g (Sigma cat. no. P5912) is placed directly into theultracentrifuge tube. This is performed by means of a small homemade funnelmade from a small sheet of flexible plastic, containing an opening small enoughto fit into the neck of the ultracentrifuge tube.4. Sodium chloride solution (d = 1.006g/mL: 0.196 molal — made by adding 11.42 g to1 L deionized water). This solution is stored at 4°C and is stable for up to 1 mo.5. Phosphate-buffered saline (PBS), pH 7.4, 0.01 M. This solution is stored at 4°Cand is stable for up to 1 mo.6. Peristaltic pump for overlaying NaCl solution onto density adjusted plasma. Weuse a Gilson Miniplus 2 (Gilson Medical Electronics, France, cat. no. 50482) at asetting of 400 for this procedure. The pump is fitted with narrow-bore AutoAnalyser tubing (Bran and Luebbe, Germany, cat. no. 116-0549-08) or similar,connected to a 21 gauge needle (21G) attached using parafilm.7. Beckman tabletop ultracentrifuge (TL100), together with a Beckman fixed-anglerotor 30° (TL100.3). The advantage of using a benchtop ultracentrifuge of this type isthat it allows rapid isolation of LDL, greatly shortening the overall length of theprocedure and allowing the isolation to be carried out in the absence of EDTA.8. Beckman Tube Topper Sealer, which includes tube topper, stand, seal cap, sealguide, heat sink, and removal tool (Beckman cat. no. 348137) together with tubespacers (Beckman cat. no. 355937). The latter are required to prevent distortionor movement of the ultracentrifuge tubes within the rotor.9. 21G needle, 2-mL syringe (clean needle and syringe for each LDL sample to beisolated, plus two extra needles per ultracentrifugation run).10. 2-mL O-ring tubes for collection of crude LDL (Sarstedt cat. no. 72.694), 10-mLtubes for collection of desalted PD10-treated LDL (Sarstedt cat. no. 57.469), and4-mL tubes for protein estimation and standard curve (Sarstedt cat. no. 55.478).11. PD10 columns containing Sephadex G25 (Pharmacia, Milton Keynes, UK). Thesecolumns are disposable. However, they can be reused many times by ensuringthey are thoroughly washed with at least 25 mL of PBS immediately prior toreuse. Columns are prepared and stored at 4°C.12. Bovine serum albumin (BSA) solution, 25 µg/mL (Sigma cat. no. A2153) (seeNote 2).13. Bio-Rad dye reagent, 300 µL/sample (Bio-Rad 500-006, Bio-Rad Hemel,Hempstead, UK), stored at 4°C.14. Disposable semimicro cuvets, 1 mL (Sarstedt, cat. no. 67.746).15. Semimicro quartz cuvets (cells), 1 mL (Starna, Romford, Essex). The number ofquartz cells required depends on the capacity of the spectrophotometer; in ourcase we require 6 cells.