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Updated: Oct 19, 2025
High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay
Published on: January 7, 2019
Lysis reagents, cell numbers, and calculation method influence high-throughput measurement of HDL-mediated
Johanna F Schachtl-Riess1, Stefan Coassin1, Claudia Lamina1
1Department of Genetics and Pharmacology, Institute of Genetic Epidemiology, Medical University of Innsbruck, Innsbruck, Austria.
Insights
Standardizing cholesterol efflux capacity (CEC) assays is crucial for cardiovascular disease research. This study identifies key technical factors, like lysis reagents and cell seeding, that significantly impact CEC assay reliability and reproducibility.
Area of Science:
- Biochemistry
- Cardiovascular Science
- Assay Development
Background:
- HDL-mediated cholesterol efflux capacity (CEC) is a potential biomarker for cardiovascular disease.
- Current CEC assays lack standardization, hindering large-scale studies and inter-laboratory comparisons.
Purpose of the Study:
- To systematically investigate technical variations impacting CEC assay performance.
- To identify methods for improving the standardization and reliability of CEC quantification.
Main Methods:
- Compared the time zero and per-well methods for CEC calculation in 506 samples.
- Evaluated the impact of lysis reagents (cholic acid, sodium hydroxide, Triton X-100) on CEC values.
- Assessed the effect of cell seeding variability on CEC measurements using a resazurin assay.
Main Results:
- The per-well method demonstrated lower sample rejection rates and improved intra- and inter-assay variability compared to the time zero method.
- Plate-to-plate correction using controls enhanced assay performance for both methods.
- Lysis reagent choice and cell seeding accuracy significantly influenced CEC quantification.
Conclusions:
- Lysis reagents, cell numbers, and assay setup are critical factors affecting CEC assay quality.
- Standardizing these technical aspects is essential for reliable CEC measurement in research and clinical settings.
Abstract:
HDL-mediated cholesterol efflux capacity (CEC) may protect against cardiovascular disease. However, CEC assays are not standardized, hampering their application in large cohorts and comparison between studies. To improve standardization, we systematically investigated technical differences between existing protocols that influence assay performance that have not been previously addressed. CEC was measured in 96-well plates using J774A.1 macrophages labeled with BODIPY-cholesterol and incubated for 4 h with 2% apolipoprotein B-depleted human serum. The time zero method, which calculates CEC using control wells, and the per-well method, which calculates CEC based on the actual content of BODIPY-cholesterol in each well, were compared in 506 samples. We showed that the per-well method had a considerably lower sample rejection rate (4.74% vs. 13.44%) and intra-assay (4.48% vs. 5.28%) and interassay coefficients of variation (two controls: 7.85%, 9.86% vs. 13.58%, 15.29%) compared with the time zero method. Correction for plate-to-plate differences using four controls on each plate also improved assay performance of both methods. In addition, we observed that the lysis reagent used had a significant effect. Compared with cholic acid, lysis with sodium hydroxide results in higher (P = 0.0082) and Triton X-100 in lower (P = 0.0028) CEC values. Furthermore, large cell seeding errors (30% variation) greatly biased CEC for both referencing methods (P < 0.0001) as measured by a resazurin assay. In conclusion, lysis reagents, cell numbers, and assay setup greatly impact the quality and reliability of CEC quantification and should be considered when this method is newly established in a laboratory.
