Wako cholesterol e kit

The cholesterol isolated from the rhodamine 6G stained silica gel G plates was assayed using the Wako cholesterol E-Kit (Wako Chemicals, Richmond, VA, USA) according to the manufacturer’s indications.

The cholesterol extraction, isolation, and quantification were achieved according to Quesada (Quesada et al. 2016 ). Briefly, the cholesterol extracted from Tc membrane and nAChR-DC were isolated from the rhodamine 6G stained silica gel G plates and further quantified using the Wako cholesterol E-Kit (Wako Chemicals, Richmond, VA, USA).

All animals used in the study were in C57BL/6 background. Our study used male mice unless otherwise specified. Mice were fed chow diet (Research Diet) and housed temperature-controlled room under a 12-h light/12-h dark cycle and pathogen-free conditions. Raly^flox/flox mice were generated by Cyagen using the strategy outlined in Fig. 1a. To the generated RALY liver-specific knockout mice and littermate controls, we treated Raly^flox/flox with adeno-associated virus (AAV) with TBG promoter (AAV8.TBG.Cre) or (AAV8.TBG.GFP; green fluorescent protein) purchased from Penn Vector Core. AAV administered intraperitoneal injection at dose of 5 × 10¹¹ GC per mice. Mice were euthanized 4 weeks after AAV injection. Liver tissues were frozen in liquid nitrogen and stored at −80 °C or fixed in 10% formalin. Blood was collected by retro-orbital bleeding, and the plasma was separated by centrifugation. Plasma lipids were measured with the Wako L-Type TG M kit, the Wako Cholesterol E kit. All animal experiments were approved by the UCLA Institutional Animal Care and Research Advisory Committee.

Baseline blood samples were collected via submandibular bleed, and post AngII infusion samples were collected via retro-orbital bleed. Blood was collected in CTAD:EDTA and centrifuged at 2000xg for 5 min to separate plasma. Total plasma cholesterol was measured with the Wako Cholesterol E Kit (Wako Diagnostics, Cat. #999-02601) and plasma triglycerides were measured using the Wako L-Type Triglyceride M kit (Wako Diagnostics, Cat. #994-02891), according to the manufacturer’s instructions.