Cellular cholesterol was measured using the Cholesterol-Glo assay (Promega). For each cell line (CRISPR-perturbed or drug-treated), we counted 100,000 cells, washed with 1xPBS twice and resuspended in 100 μL cell lysis solution for 30 min at 37°C. Following the incubation, 10 μL of cell lysate was used to measure the cholesterol levels in 96 well plates in a final reaction volume of 100uL per well (without esterase). Luminescence was measured after incubating the plate for 1 hour at room temperature in the dark. Using the same lysis sample, the protein concentration was determined using the BCA assay (Thermo). The cholesterol levels were normalized to the respective protein concentration of each sample. A549ACE cells were incubated with 10 μM amlodipine (Sigma) or DMSO for 24 hours. Cholesterol levels were then determined as described above.
Cholesterol glo assay
Manufactured by Promega
Sourced in United States
The Cholesterol-Glo assay is a luminescent-based detection kit that quantifies total cholesterol levels in samples. The assay utilizes a coupled enzyme reaction that produces a proportional luminescent signal to the amount of cholesterol present.
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Lab products found in correlation
3 protocols using cholesterol glo assay
1
Cholesterol Quantification in Cells
2
GRAMD1B Knockdown and Cholesterol Depletion
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On day 0, 100,000 NT or GRAMD1B shRNA SKMEL2 cells were pretreated in 4 μg/mL dox-containing media in 6-well dishes to induce GRAMD1B knockdown for 48 hours. On day 2, cells were switched to either dox-containing control media (DMEM, 10% FBS, 1% antibiotics, 4 μg/mL dox) or cholesterol depletion media (DMEM, 10% lipoprotein-free FBS, 1% antibiotics, 10 μmol/L simvastatin, and 200 μmol/L mevalonic acid, 4 μg/mL dox) for another 48 hours. Because the mevalonate pathway is critical for cell viability, mevalonate supplementation was included in the cholesterol depletion mixture as done in Trinh and colleagues (121 (link)). On day 4, cholesterol depletion was confirmed through Promega's cholesterol Glo assay, and cells were seeded for invasion assays.
3
Cellular Cholesterol Quantification
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Total cellular cholesterol levels were measured using the Cholesterol Glo™ Assay (J3190, Promega, USA) according to the manufacturer’s description. A total of 5000 cells was used and the H151 (1 µM) condition was incubated for 1 h with the compound before lysis.
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