title: How to use Nile Red, a selective fluorescent stain for microalgal neutral lipids.
abstract: The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125mg/mL), or EDTA (3.0 to 3.8mg/mL). Temperatures between 30 and 40°C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25μg/mL and 2.0μg/mL, and the cell concentration >5×10(4)cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.
authors.affiliation: Escuela de Ingeniería y Ciencias, Tecnologico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501, Monterrey 64849, N.L., Mexico.
authors.forename: Gibrán S
authors.initials: GS
authors.lastname: Alemán-Nava
authors.affiliation: Laboratory Aquatic Biology, KU Leuven Kulak, E. Sabbelaan 53, Kortrijk, Belgium.
authors.forename: Sara P
authors.initials: SP
authors.lastname: Cuellar-Bermudez
authors.affiliation: Algal Biotechnology Group, Centro de Investigación y Desarrollo de Recursos y Tecnologías Agroalimentarias (CIDERTA), Huelva University, Marine International Campus of Excellence (CEIMAR), Huelva, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Cuaresma
authors.affiliation: Wageningen University, Bioprocess Engineering, AlgaePARC, P.O. Box 16, 6700 AA, Wageningen, The Netherlands.
authors.forename: Rouke
authors.initials: R
authors.lastname: Bosma
authors.affiliation: Laboratory Aquatic Biology, KU Leuven Kulak, E. Sabbelaan 53, Kortrijk, Belgium.
authors.forename: Koenraad
authors.initials: K
authors.lastname: Muylaert
authors.affiliation: Escuela de Ingeniería y Ciencias, Tecnologico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501, Monterrey 64849, N.L., Mexico; Swette Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, PO Box 875701, Tempe, AZ 85287-5701, USA.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Ritmann
authors.affiliation: Escuela de Ingeniería y Ciencias, Tecnologico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501, Monterrey 64849, N.L., Mexico. Electronic address: r.parra@itesm.mx.
authors.forename: Roberto
authors.initials: R
authors.lastname: Parra
mesh_terms: D002138:Calibration; D002473:Cell Wall / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry; D008055:Lipids / Q000032:analysis*; D058086:Microalgae / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry*; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D008055:Lipids; D010078:Oxazines; D004492:Edetic Acid; C044808:nile red; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Biodiesel; Microalgae; Neutral lipids; Nile Red; Protocol