Article: Bono, MS; Garcia, RD; Sri-Jayantha, DV; Ahner, BA; Kirby, BJ; (2015) “Measurement of Lipid Accumulation in Chlorella Vulgaris Via Flow Cytometry and Liquid-State H-1 NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol “, PLOS ONE, 10 (8)
Abstract: In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state H-1 NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative H-1 NMR.
Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r(2) = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.
Funding Acknowledgement: Empire State Stem Cell Fund through New York State Department of Health Contract [C026718]; US Department of Defense (US DoD) through the National Defense Science and Engineering Graduate (NDSEG) Fellowship program; National Institutes of Health (NIH) via the Physical Sciences-Oncology Center at Cornell University [U54CA143876]
Funding Text: This work made use of the Cornell University NMR Facility and the Cornell University Flow Cytometry Core Laboratory. The Flow Cytometry Core Laboratory acknowledges support by the Empire State Stem Cell Fund (stemcell.ny.gov) through New York State Department of Health Contract #C026718. Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Board, the New York State Department of Health, or the State of New York. MSB was supported by the US Department of Defense (US DoD) through the National Defense Science and Engineering Graduate (NDSEG) Fellowship program (ndseg.asee.org) and received partial support from the National Institutes of Health (NIH) via the Physical Sciences-Oncology Center at Cornell University (Award U54CA143876, physics.cancer.gov).