Cholesterol quantitation kit

A cholesterol quantitation kit (Sigma-Aldrich, USA) was used to determine the cholesterol content of podocytes. Cells (1 × 10⁶) were extracted with 200 μL of chloroform:isopropanol:IGEPAL CA-630 (7:11:0.1) in a microhomogenizer. The samples were centrifuged at 13000 g for 10 min to remove insoluble material. Next, the samples were transferred in the organic phase to a new tube and air dried at 50 °C to remove any residual organic solvent. Dried lipids were dissolved in 200 μL of the cholesterol assay buffer and then vortexed until the mixture was homogenous. The reaction mixtures were prepared according to the kit instruction, and the cholesterol content of the lipid solution was determined using a fluorometric assay. The fluorescence intensity was analyzed in a fluorescence microplate reader with an excitation wavelength of 535 nm and an emission wavelength of 587 nm.

One percent (1%) of sub-cultured Lactobacillus isolates were inoculated into MRS broth (10 ml) containing 0.3% (b/v) ox gall (Oxoid; LP0055) and 100 mg/L water-soluble cholesterol (Sigma Aldrich; C4951) to prepare cholesterol removal assay by growing cells. For resting cell preparation, 1% of sub-cultured Lactobacillus isolates that were previously washed 3 × with PBS were suspended in 0.05 M phosphate buffer (pH 6.8) supplemented with 0.3% (w/v) ox gall and 100 mg/L cholesterol. Heat-killed cells were prepared from saline-suspended cell pellets of sub-cultured Lactobacillus isolates that were heated at 121°C for 15 min before being added into MRS broth containing 0.3% ox gall and 100 mg/L cholesterol. Incubation was done at 37°C for 20 h in all assays, after which the mixture was centrifuged at 13,000 rpm for 5 min at 4°C. MRS broth with and without the addition of 0.3% ox gall (b/v) and 100 mg/L water-soluble cholesterol were used as a negative and positive control, respectively. The remaining cholesterol in the supernatant was measured using Cholesterol Quantitation Kit (Sigma Aldrich; MAK048).

To uncover the ameliorative ability of Ginkgolide B on cholesterol accumulation in ECs, above HUVECs were collected and their total cholesterol (TC) and free cholesterol (FC) were quantified by Cholesterol Quantitation Kit supplied by Sigma-Aldrich, USA.

To explore the effect of QCT on cholesterol accumulation in macrophages, murine RAW264.7 cells were treated with ox-LDL (20 μg/ml) and QCT (20 μM) as the method described above. Cells were collected and processed for cholesterol extraction (Cholesterol Quantitation Kit from Sigma-Aldrich, MAK043) according to the instruction (Kain et al., 2015 (link)). Total or free cholesterol concentration of samples was estimated using commercial Cholesterol Quantitation Kit (Sigma-Aldrich, USA) by the automated analyzer according to the instruction and previous report. Briefly, 50 μl of the diluted standards and samples were incubated with 50 μl of reaction mixes containing cholesterol assay buffer, cholesterol probe, cholesterol enzyme mix, cholesterol esterase for 60 min at 37°C. After incubation, the absorbance was measured at 570 nm on a multiscan spectrum (EnSpire Multimode Plate Readers, PerkinElmer) and the cholesterol standard curve (0, 20, 40, 60, 80, 100 μg/ml) was generated using the standard cholesterol solutions. All standards and samples were run in duplicate.

To quantify the cholesterol concentration in distal jejunum and descending colon, a Cholesterol Quantitation Kit (Catalog Number: MAK043, Sigma-Aldrich, St. Luis, MO, USA) was used for lipid extraction and quantitation by fluorometric detection following manufacturer’s instruction. Prior to cholesterol extraction, adipose tissues were removed manually using a scalpel, as the study of lipid storage was not the objective of this study, and the rest of the tissues were ground and mixed. Approximately 100 mg of tissue was used for cholesterol extraction and measurement according to the kit instructions, and the fluorescence intensity was detected at: λ_ex = 535 and λ_em = 587 nm. Two tailed sample t.test (significance level: 0.05) was performed to determine whether cholesterol content of the distal jejunum and descending colon differed between SS and NS.