Cholesterol rtu kit

Cholesterol binding (CH_b) activity was determined on the basis of its loss after incubation with FOCE-LGG and FOCE-LGG-110, FOCE-LGG -140 and FOCE-LGG -170 samples obtained after passage through SHIME. Samples taken from Reactor 3 as well as from “fresh” FOCE-LGG were loaded into 12-well plates with 1% cholesterol (dissolved in 96% ethanol) to obtain a final cholesterol concentration of 1.66 g/L. The samples were incubated for 18 h (30 °C), then centrifuged (Centrifuge MPW 351R, 7500 rpm, 10 min, 4 °C) to separate the culture fluid. In order to determine the cholesterol residues in the samples, the procedure of Cholesterol RTU kit (BioMerieux, Marcy l’Etoile, France) was followed. The absorbance measurement was performed using the NanoDrop ND 1000 spectrophotometer at 500 nm. The percentage of bound cholesterol from the environment was calculated according to the formula: CHb=[(A−B)×100]A
where: A is the initial absorbance of mixture, B is the absorbance of mixture after inxubation.

To assess plaque size and composition, aortic root cryosections were stained with Oil red O, hematoxylin and eosin, picrosirius red (All Sigma) or with anti-α–smooth muscle actin (Sigma), anti-CD3 (Dako) or MOMA2 (Biorad) antibodies. Images were analyzed using ImageJ (National Institutes of Health). Cellularity was determined by quantifying the proportion of nuclear-stained area within plaques (Hoechst staining). Serum total and oxidation-specific epitope antibodies were analyzed using kits from Bethyl labs or as previously described.^{28 (link)} Total cholesterol was quantified using a cholesterol RTU kit (Biomerieux) and serum BAFF by ELISA (BioTechne).