Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

Abstract

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content. ' 2012 International Society for Advancement of Cytometry

Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica

“…Lipid composition was verified using confocal microscopy and BODIPY © 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4bota-3a,4a-diaza-s-indacene), a neutral lipid stain superior to Nile Red in certain systems [14], [15]. To verify that bodies observed in both light and electron microscopy were in fact lipid droplets (LDs), digestive diverticula were dissected from adult E. chlorotica tissue, stained with 1 µg ml −1 final concentration of BODIPY 505/515, and visualized on a Nikon A1R confocal microscope.…”

Imaging the accumulated intracellular microalgal lipids as a response to temperature stress

“…To identify algae species with high lipid content, lipophilic fluorescent dye Nile red which is commonly used as a vital stain for lipid-containing vesicles [also referred to as lipid bodies (LB)] was used within algal cells (Bertozzini et al 2011;Chen et al 2009;Cirulis et al 2012;Cooper et al 2010;Eibl et al 2014;Greenspan and Fowler 1985;Siaut et al 2011;Wang et al 2009). For the microscopy study, cells were grown to stationary phase.…”

Screening native isolates of cyanobacteria and a green alga for integrated wastewater treatment, biomass accumulation and neutral lipid production

“…The BODIPY fluorescence signal was collected using a 530/30 nm band-pass filter and data collected using FACSDiva software v. 6.1.3 prior to analysis using Flowing software v.2.5 (http://www.flowingsoftware.com/). The gating strategy employed was based on that of [28] using fluorescence in the PE-Cy7 channel (780/60 nm band-pass filter) versus Chl autofluorescence in the PerCP-Cy5-5 channel (695/40 nm band-pass filter) to remove non-cell signals from the population of interest. Unstained cell samples and a cell free sample (to which BODIPY was added) were used as controls.…”