Lysis reagents, cell numbers, and calculation method influence high-throughput measurement of HDL-mediated cholesterol efflux capacity

HDL-mediated cholesterol efflux capac-ity (CEC) may protect against cardiovascular disease. However, CEC assays are not standardized, hampering their application in large cohorts and comparison between studies. To improve standard-ization, we systematically investigated technical differences between existing protocols that influ-ence assay performance that have not been previ-ously addressed. CEC was measured in 96-well plates using J774A.1 macrophages labeled with BODIPY-chole sterol 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 inter-assay 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 differ-ences 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 reli-ability of CEC quantification and should be considered when this method is newly established in a laboratory.

Automated summary

The study found that the per-well method for measuring CEC had lower rejection rates and coefficients of variation compared to the time zero method, and the use of four controls on each plate improved assay performance for both methods. Additionally, the choice of lysis reagents, cell numbers, and assay setup significantly impact the quality and reliability of CEC quantification.