Cells were seeded in 6-well plates at a density of 1 · 106 cells/well, polarized for 18 hours, and incubated or not with LDLs for 24 hours. After this incubation, cholesterol was extracted from cells by adding 200 μL of chloroform : isopropanol : IGEPAL CA-630 (7 : 11 : 0.1) in a microhomogenizer. The solution was then centrifuged at 13 000 ×g for 10 minutes in order to remove insoluble material. The organic phase was transferred in a new tube, air-dried at 50°C to remove chloroform, and put under vacuum for 30 minutes to remove any residual organic solvent. The dried lipids were dissolved with 220 μL of the cholesterol assay buffer and were vortexed until the lipid solution was homogeneous. Cholesterol was finally quantified following the manufacturer's instructions by using reaction mixes containing the cholesterol assay buffer, probe, enzyme mix, and/or cholesterol esterase. The absorbance was measured at 570 nm (Cholesterol Quantitation Kit MAK043) (Sigma-Aldrich, St. Louis, MO, USA) and results were expressed as μg/μL of free, esterified, or total cholesterol following the manufacturer's instructions.
Cholesterol quantitation kit mak043
Manufactured by Merck Group
Sourced in Italy, United States
The Cholesterol Quantitation Kit-MAK043 is a laboratory assay designed to quantify the total cholesterol levels in a sample. The kit utilizes an enzymatic reaction to produce a colorimetric signal, which can be measured using a spectrophotometer. The intensity of the color produced is proportional to the amount of cholesterol present in the sample. The kit provides the necessary reagents and protocols to perform the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Spark microplate reader, by Tecan (1 mentions)
Cobas integra 400 plus, by Roche (1 mentions)
Free fatty acid quantitation kit mak044, by Merck Group (1 mentions)
Triglyceride determination kit tr0100, by Merck Group (1 mentions)
Phospholipid assay kit mak122, by Merck Group (1 mentions)
Dialysis tubing cellulose membrane, by Merck Group (1 mentions)
Matrigel basement membrane matrix 356234, by Corning (1 mentions)
Millipore simplicity 185 ultrapure water system, by Merck Group (1 mentions)
A549 atcc ccl 185, by American Type Culture Collection (1 mentions)
Hepg2 atcc hb 8065, by American Type Culture Collection (1 mentions)
6 protocols using cholesterol quantitation kit mak043
1
Cholesterol Quantitation Assay Protocol
2
Quantification of Tissue and Serum Cholesterol
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The cholesterol amount in tissues and serum were measured by using the Cholesterol Quantitation Kit-MAK043 (Sigma-Aldrich, Milan, Italy), following the manufacturer’s instructions. Briefly, 10 mg of hepatic tissues and of each brain area were lysed in 200 µL of chloroform:isopropanol:nonylphenylpolyethylene glycol solution (Nonidet P-40) (7:11:0.1). The extracted lipids were resuspended in buffer solution and 5 µL were used for each sample. For the serum cholesterol measurement, 2 µL of the sample were directly added to the reaction mix. The assay detects total cholesterol (cholesterol and cholesteryl esters) when cholesterol esterase is included in the reaction, or free cholesterol when it is not included. The amount of cholesteryl ester can be determined by subtracting the value of free cholesterol from the total (cholesterol plus cholesteryl esters). The enzymatic assay results in a colorimetric product, proportional to the cholesterol present in the sample. The amount of cholesterol present in the samples was revealed by determining the absorbance at 570 nm with the Tecan Spark microplate reader (Männedorf, Switzerland). All samples were run in duplicate.
3
Quantification of Lipid Levels
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Triglycerides and total-, LDL- and HDL-cholesterol were measured by means of an enzymatic method (Cobas Integra 400 Plus, Roche Diagnostics, Rotkreuz, Switzerland), with intra- and inter-assay coefficients of variation < 2%. The cholesterol amount in brain tissue was quantified by the Cholesterol Quantitation Kit-MAK043 (Sigma Aldrich, Milan, Italy) following the manufacturer’s instructions.
4
Cholesterol Quantification in Tissue Samples
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The cholesterol amount in tissue samples was measured using the Cholesterol Quantitation Kit-MAK043 following the manufacturer’s instructions (Sigma Aldrich, Milan, Italy).
5
Lipid Analysis in Rat Serum and Tissues
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Serums from each rat were centrifuged at 825 g for 20 min and 4 °C to determine lipid ratios. Stored at −80 °C until the analysis. Total cholesterol (Sigma-Aldrich Cholesterol Quantitation Kit MAK043), triglyceride (Sigma Serum Triglyceride Determination Kit TR0100), phospholipid (Sigma-Aldrich Phospholipid Assay Kit MAK122), and free fatty acid (Sigma-Aldrich Free Fatty Acid Quantitation Kit MAK044) analysis were performed in each serum sample.
Liver and kidneys were stored at −80 °C in phosphate buffer (1/10 w/v) to determine total lipid amounts. The liver and kidneys in phosphate buffer (20 mL) were put into an extraction thimble that contained chloroform/methanol (40 mL; 3:2 v/v), separately. The obtained mixture was homogenized at 155 rpm for 15 min. Then, water (8 mL) was added, and the mixture was shaken vigorously to facilitate the transfer of oil into the chloroform and other products into the water–methanol layer. The chloroform layer was then separated via a separation funnel and collected. The extraction steps were repeated two times. The obtained chloroform layers were combined and evaporated using an evaporator (65 °C, for 20–30 min.). Then, the trace amount of chloroform was evaporated in drying oven and weighed remaining oil [59 (link)].
Liver and kidneys were stored at −80 °C in phosphate buffer (1/10 w/v) to determine total lipid amounts. The liver and kidneys in phosphate buffer (20 mL) were put into an extraction thimble that contained chloroform/methanol (40 mL; 3:2 v/v), separately. The obtained mixture was homogenized at 155 rpm for 15 min. Then, water (8 mL) was added, and the mixture was shaken vigorously to facilitate the transfer of oil into the chloroform and other products into the water–methanol layer. The chloroform layer was then separated via a separation funnel and collected. The extraction steps were repeated two times. The obtained chloroform layers were combined and evaporated using an evaporator (65 °C, for 20–30 min.). Then, the trace amount of chloroform was evaporated in drying oven and weighed remaining oil [59 (link)].
6
Amphiphilic Cyclodextrin Synthesis and Characterization
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Nonionic amphiphilic CDs 6OCaproβCD (Mw: 1822 g/mol) [37] and6OCaproαCD (detailed synthesis and characterization data were given in the Supplemental file) (Mw: 1562 g/mol) and polycationic amphiphilic CD PC βCDC6 [38] (Mw: 3178 g/mol) were synthesized and purified according to previously reported procedure. Methyl-βCD (Cavasol® W7 M Pharma) was purchased Wacker Chemie AG, USA. Erlotinib hydrochloride (Mw: 429.9 g/mol) was a kind gift of Nobel Pharmaceuticals, Turkey purchased from Hetero Labs (Gaddapotharam, India). Dialysis Tubing Cellulose Membrane (avg. flat width 25mm, MWCO: 14000 Da) was purchased from Sigma &Aldrich, Germany. A549 (ATCC® CCL-185™) and HepG2 (ATCC® HB-8065™) cell lines were purchased from American Type Culture Collection (ATCC). Poly(2hydroxyethyl methacrylate) (poly-HEMA) (P3932) was purchased from Sigma-Aldrich, Germany. Matrigel® Basement Membrane Matrix (356234) was purchased from Corning, USA. Cholesterol quantitation kit (MAK043) was purchased from Sigma-Aldrich, USA. All other chemicals used were of analytical grade and obtained from Sigma & Aldrich, USA. Ultrapure water was obtained from Millipore Simplicity 185 Ultrapure Water System (Millipore, France).
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