Beckman ls 6500

Mice were deprived of food overnight prior to the evaluation of intestinal Ca absorption. On the morning of the test, mice were anesthetized with a cocktail of ketamine (22 mg/mL) and xylazine (33 mg/mL) (0.1 mL/20 g body weight). Ca absorption was examined by an oral gavage method originally reported by Van Cromphaut et al. [16 (link)] and used by us elsewhere [12 (link)]. Briefly, mice were given an oral gavage of a solution containing 0.1 mmol/L CaCl₂, 125 mM NaCl, 17 mM Tris Base, enriched with 20 μCi ⁴⁵CaCl₂/ml (Perkin Elmer, Waltham, MA), and containing 25 mmol/L of either glucose or fructose, (10 L of buffer per g of body weight) (n=9-12 mice from each diet group per buffer). Blood was collected in live anesthetized animals 10 minutes after administration of the dosing solution using the GoldenRod lancet (Medipoint, Inc., Mineola, NY) to puncture the submandibular vein. Serum was isolated (10 μL) and bleached for 30 min, pH was neutralized and samples were analyzed using a liquid scintillation counter (Beckman LS 6500; Beckman Coulter Inc., Fullerton CA) for 1 min. The change in the serum calcium concentration was calculated from the ⁴⁵Ca content of the serum and the specific activity of the administered calcium.

Ac-LDL loading and cholesterol efflux of VSMCs were measured using a
method similar to that used for macrophage cholesterol efflux measurement
[28 (link)]. Briefly, rat
VSMCs were cultured in 24-well plates in DMEM containing 10% FBS till
confluence and further cultured in DMEM containing 0.25% FBS overnight,
followed by loading with 50 μg/ml ac-LDL incorporated with
³H-cholesterol with or without LPS (10 ng/ml) and SsnB (10
μM) for 24 h. Then efflux medium (DMEM containing 20 μg/ml
apoAI) was added to initiate cholesterol efflux for 24 h. ³H
radioactivity in efflux medium and cells were counted using a Beckman LS6500
liquid scintillation counter (Beckman Coulter, Indianapolis, IN). Cellular
protein was determined using Lowry assay. Total cholesterol loading and
cholesterol efflux efficiency were calculated.

Mice brains were homogenized in buffer (4 mM HEPES, 0.32 M sucrose, pH 7.4) and centrifuged at 1000 g for 10 min. The supernatant was centrifuged at 20,000 g for 20 min and the pellet was resuspended in 1.6 ml of 0.32 M sucrose. After homogenization in 6.4 ml of the cold distilled water by a glass/Teflon homogenizer, 1 ml of buffer containing 250 nM HEPES and 1 M potassium tartrate was added and the mixture was centrifuged at 20,000 g for 20 min. The supernatant was centrifuged again at 120,000 g for 2 h and the vesicles were resuspended in 1 ml of buffer (100 mM potassium tartrate, 25 mM HEPES, 0.1 mM EDTA, 0.05 mM EGTA, and 1.7 mM ascorbate, pH 7.4). 300 ml of vesicle solution including NE was incubated for 30 min at 30°C with APOE3 or APOE4 recombinant proteins, and then a tracer of 2% [³H] NE was added and incubated for 5 min at 30°C. After termination of the reaction by addition of 5 ml of cold assay buffer, the vesicles were filtered through the membrane filters (Millipore). The filters were placed in scintillation fluid and counted by Beckman scintillation counter (Beckman LS6500).

Macrophage cultures were metabolically labeled with [³H]-glucosamine (50 µCi/ml) in the presence or absence of 100 ng/ml LPS and/or 0.5mMchloroquine for 2, 4, 6 and 24 h. Media and cell layer fractions were harvested separately and digested with pronase (100 µg/ml) in 0.5 M Tris pH 6.5 overnight at 37 °C. Following digestion, the pronase was inactivated by heating to 100 °C for 20 min. Radiolabeled macromolecules were then recovered and separated from unincorporated precursor by precipitation on nitrocellulose membranes using slot blot analysis (Agren et al., 1994 (link)). Briefly, 200 µl of sample was added to an equal volume of 2% cetylpyridinium chloride, 50 mM NaCl (CPC wash solution) and the solution blotted onto 0.45 µm nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membrane was washed six times in CPC wash solution and once in deionized water before air-drying at room temperature overnight. Incorporation of [³H]-glucosamine into hyaluronan was measured by digesting an equivalent radiolabeled aliquot with Streptomyces hyaluronidase (2 U/ml) for 24 h at 37 °C before slot blotting. Hyaluronan was measured as the amount of hyaluronidase-sensitive material precipitated to the nitrocellulose membrane. All scintillation counting was done on Beckman LS 6500 (Beckman Instruments, Fullerton, CA).