Membrane pellets were obtained from neurons cultured in 60-mm petri dishes. The cells were lysed in MES buffer (25 mM, pH 7.1), and the membrane pellet of the neurons was obtained by ultracentrifugation of the extract at 70000 rpm (TLA 100.1 rotor, Beckman) for 1 hour at 4 °C. Quantification of cholesterol concentration was performed using the Amplex Red cholesterol assay (Invitrogen- A12216). Fluorescence levels in the final AmplexRed–membrane pellet solution were measured using a Tecan spectrophotometer.
Amplex red cholesterol assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Amplex Red Cholesterol Assay is a fluorometric assay used to detect and quantify cholesterol levels. It measures the amount of cholesterol present in a sample by monitoring the oxidation of Amplex Red reagent.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Subcellular protein fractionation assay, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Tla100.1 rotor, by Beckman Coulter (1 mentions)
Labassay phospholipid, by Fujifilm (1 mentions)
Fluoroskan plate reader, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Filipin staining, by Merck Group (1 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Immobilon p pvdf, by Merck Group (1 mentions)
Streptavidin conjugated agarose beads, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
46 protocols using amplex red cholesterol assay
1
Cholesterol Quantification in Neuronal Membranes
2
Quantifying Cholesterol in Drosophila
3
Cholesterol Quantification in Rab7 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
22L-N2a cells transfected for 48 h with pEGFP-Rab7 plasmids expressing EGFP-WT Rab7, EGFP-Rab7-Q67L, and EGFP-Rab7-T22N and untransfected control N2a cells were used. The cells were lysed as described (34 ) and centrifuged. The postnuclear supernatant was used for the Amplex red cholesterol assay (Invitrogen), bicinchoninic acid assay analysis, and immunoblotting to determine the transfection efficiency. Briefly, the total cholesterol levels were quantified as recommended by the manufacturer’s instructions and were normalized to the respective protein concentrations of the samples.
4
Quantification of Cellular Cholesterol and Phospholipids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One million cells were plated in 150‐mm petri dishes 24 h before the experiment. Cells were treated with 10 mM methyl‐β‐cyclodextrin (MβCD, C4555 Sigma) for 30 min for the positive control. Cells were washed three times and scraped in 500 μl of PBS. Cells in PBS (500 μl) were mixed with 2 ml of EtOH/chloroform (2/1) solution in glass tubes. After 5 min of centrifugation at 1,400 g, the supernatant was transferred into a new tube. Then, 0.5 ml of 50 mM citric acid, 1 ml of water, and 0.5 ml of chloroform were added. After 20 min of centrifugation at 1,400 g, the lower phase‐containing lipids were transferred in new glass tubes and dried in a stream of nitrogen. For the lipids quantification, dried lipids were solubilized in EtOH 95% solution. Cholesterol quantification was performed with the Amplex Red cholesterol assay (A12216 Invitrogen), and phospholipids quantification was performed with LabAssay phospholipid (296‐63801 Wako Chemicals) according to the manufacturer instructions.
5
Macrophage Cholesterol Metabolism Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thioglycollate-elicited peritoneal macrophages (2x106 cells) isolated from macLRP1-/- and LRP1+/+ mice were plated in 100 mm dishes in serum free media for 3 h at 37°C. Cells were scraped off the plate into 4 ml PBS and centrifuged at 1000 rpm for 5 min. Cell pellets were extracted with 300 μl isopropanol with sonication (2 x 10 s, medium setting). The cell extract was centrifuged at 12000 rpm for 5 min and the supernatant was removed and assayed for cholesterol. Total cholesterol and free cholesterol was determined using the Invitrogen Amplex Red Cholesterol Assay in the presence or absence of cholesterol esterase respectively. Samples were diluted 10- to 40-fold before assaying. The cell pellet was taken up in 50 μl 4M guanidinium hydrochloride and the protein concentration determined using the Pierce BCA assay.
6
Cholesterol Quantification using Amplex Red
7
Quantification of Intracellular Cholesterol and Secreted PSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Amplex Red Cholesterol assay (Invitrogen) was performed as per manufacturer’s instructions to measure the total intracellular cholesterol amount. 50 μL of cell extracts and cholesterol reference standards were aliquoted in triplicates into a 96-well plate and Amplex Red reaction buffer and working solution were added. Plates were incubated for thirty minutes at 37°C in the dark. Fluorescence was measured using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cholesterol concentration was normalized to the amount of protein present in the lysates.
The amount of PSA secreted by the cancer cells was quantified using the PSA ELISA assay (ProClin International). 50uL of reference standards and media from each treatment and control wells were aliquoted in triplicates into a 96-well plate. Following incubations with reaction buffer, enzyme conjugate reagent, and TMB reagent, the optical density was measured at 450 nm and the PSA concentration was normalized to the amount of protein present in the media.
The amount of PSA secreted by the cancer cells was quantified using the PSA ELISA assay (ProClin International). 50uL of reference standards and media from each treatment and control wells were aliquoted in triplicates into a 96-well plate. Following incubations with reaction buffer, enzyme conjugate reagent, and TMB reagent, the optical density was measured at 450 nm and the PSA concentration was normalized to the amount of protein present in the media.
8
Cholesterol Quantification in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cholesterol levels in whole‐cell lysates were calculated by an Amplex Red cholesterol assay (Invitrogen). Briefly, samples were diluted in reaction buffer, and an equivalent volume of Amplex Red working solution (300 × 10−6m Amplex Red, 2 U mL−1 cholesterol oxidase, 2 U mL−1 cholesterol esterase, and 2 U mL−1 horseradish peroxidase) (Thermo Fisher Scientific, A12216) was added. Samples were incubated at 37 °C for 30 min, and the fluorescence was measured at EX:560 nm and Em:590 nm using a microplate reader. Cholesterol values were calculated using known cholesterol solutions and normalized to the protein content as measured by a Bradford Protein Assay Kit (Bio‐Rad).
9
Cholesterol Quantification in Placental Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Folch extraction was performed on 70 μg of protein from PHT lysate as described38 (link). Lysates were incubated with methanol/chloroform (1:2 v/v, 30 min, 50 °C). Then, 1 volume of water was added (18 h, 4 °C), and the reaction was centrifuged (750 RCF, 20 min, 4 °C). The methanol/water phase was removed, and the chloroform phase containing cellular cholesterol was recovered and completely evaporated under nitrogen. Cholesterol extracted from PHT was determined with the Amplex Red Cholesterol assay (Invitrogen, USA) in the presence or absence of the enzyme cholesterol esterase for determination of total (TC) and free cholesterol (FC), respectively. Cholesterol esters were determined as the difference between TC and FC. FC was also determined by Filipin staining (Sigma-Aldrich, USA) as described39 (link). PHT cells were fixed with 4% paraformaldehyde, their autofluorescence was quenched with glycine in PBS (1.5 mg/mL, 20 min, 20 °C), and then they were incubated with Filipin (25 μg/mL, 30 min, 20 °C). Images were obtained in an EVOS FL Imaging System (Life Technologies, USA), and the fluorescence was quantified with ImageJ version 1.48 (NIH, USA).
10
Quantitative Cholesterol Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplex Red cholesterol assay was performed (according to manufacturer instructions; Invitrogen) to determine the abundance of cholesterol. Each reading was normalized to protein concentration (determined by bicinchoninic acid (BCA) assay) in the same samples. Technical triplicates were measured for each sample. Shown are values normalized to untreated cells. Reported are the average and standard deviations from at least three independent biological replicates.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!