Live imaging fluorescent dye for lipid droplets (LDs) LipiDye^™ II

LipiDye^™ II is a highly sensitive lipid droplets staining reagent for long-term live cell imaging. In addition to its high lipid droplets specificity, LipiDye^™ II shows low toxicity and extremely high photostability, making it useful for long-term observation over several days, live cell imaging of lipid droplet fusion and degradation processes, and visualization of small LDs using STED super resolution microscopy.

Lipid droplets (LDs) are organelles that are historically found in adipocytes and have a unique phospholipid monolayer and store neutral lipids such as triglycerides and sterol esters (Below figure left). LDs are thought to act as intracellular neutral lipid storage organs, and are often reported to be associated with obesity and diseases. In recent years, it has been reported that LDs are found not only in adipocytes but also in various other cell types, including hepatocytes, smooth muscle cells, and glial cells, and have various functions, such as metabolic control and regulation of gene expression, as well as their role as storage organs for neutral lipids, as classically known. LDs have been observed in a variety of cells, and it is known that LDs in non-adipocytes are less than 1 μm in diameter and much smaller than those in adipocytes (10-100 μm). The imaging reagent for the observation of small LDs in non-adipocyte has been expected, since existing reagents such as Nile Red stain other than LDs (low signal-to-noise ratio) and are unsuitable for live cell imaging and the observation method of small LDs has been limited to electron microscopy.

LipiDye^™ II

Staining of various cells
3T3-L1, HepG2, COS-7 and HeLa cells were stained with LipiDye^™ II (1 µM) and observed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). In the case of HepG2, cells were pretreated with fatty acids for one day before LipiDye^™ II staining. In HeLa cells, small LDs of approximately 1 µM were clearly observed. (Scale bar: 20 µm, HeLa cell enlarged 5 µm).

Multicolor imaging with ER marker
COS7 cells expressing ER-resident red fluorescent protein (ER-mKO1) were stained with LipiDye^™ II (1 uM) and observed by confocal microscopy (LipiDye^™ II; Ex. 473 nm/Em 490-540 nm, ER-mKO1; Ex. 635 nm/Em 660-710 nm). Small LDs less than 1 um were frequently observed in the network structure of ER. (Scale bar: 20 um, 5 um and 1 um). This result correlates well with the fact that LDs are biosynthesized from the ER.

Long-term staining during adipocyte differentiation and maturation
Lipid droplet maturation of 3T3-L1 preadipocytes induced to differentiate into adipocytes was observed by confocal microscopy using LipiDye^™ II (1 uM) for 8 days (excitation 473 nm / fluorescence 490-540 nm). 3T3-L1 cells were cultured in differentiation medium containing LipiDye^™ II (1 uM) for 2 days and then the medium was replaced with maintenance medium containing LipiDye^™ II (1 uM) every 2 days. The adipocyte maturation process could be observed even after long-term treatment with LipiDye^™ II without any cytotoxicity or effect on adipocyte differentiation.

Dynamic behavior analysis of newly formed LDs by live cell imaging
Newly formed LDs of 3T3-L1 preadipocytes during induction of differentiation into adipocytes were observed by long time-lapse imaging (Z-stack imaging) for about 24 hours (confocal microscopy: excitation 473 nm / fluorescence 490-540 nm, 20 z-images/10 min). Time-lapse imaging was started immediately after the addition of differentiation medium (containing LipiDye^™ II) to 3T3-L1 previously stained with LipiDye^™ II. After ~10 hours differentiation, small LDs were observed (650 min, white arrows) and some LDs were docking with other LDs during adipogenesis (950-1450 min, yellow arrow). (Scale bar; 1 um).

Time-lapse Z-stack imaging of lipolysis and lipogenesis
After the differentiated 3T3-L1 cells were treated with forskolin (10 μM), an activator of adenylyl cyclases, and IBMX (100 nM), an inhibitor of phosphodiesterases, time-lapse Z-stack imaging (15 z-images/4 min, for 800 min) were performed by confocal microscopy (Ex. 473 nm/Em 490-540 nm) to observe the lipid droplet shrinkage process associated with triacylglycerol degradation. Some large LDs clearly contracted (white arrows) and numerous newly formed small LDs were observed (yellow arrows) in a time-dependent manner. Since the high photostability and high S/N ratio of the LipiDye^™ II allows us to observe even the smallest newly formed LDs, the increase and decrease of LDs can be quantitatively observed.

Semi-quantitative analysis of cellular lipid droplets by fluorescent plate reader
Three cell lines (human renal cancer cell line 786-O, mouse neuroblastoma cell line Neuro2a, and Chinese hamster ovarian cell line CHO) were seeded in 96 well plate at 1 x 104 cell/well and cultured in DMEM containing 10% FBS (10% FBS/DMEM). After 24 hours, cells were treated with 10-200 uM oleic acid in 10% FBS/DMEM for further 24 hours to promote growth of lipid droplets. After washing cells, the cells were stained by 5 uM LipiDye^™ II-containing 2% FBS/DMEM for 2 hours. Just before fluorescent plate leader measurement, cells were washed by PBS twice and fluorescent intensity (Ex 420 ±5 nm/Em 503 ±10 nm) of each well was measured. In all cell lines, oleic acid dose-dependency was observed.

Live-cell STED super resolution microscopy imaging of small LDs
HeLa cells were treated with 1 uM LipiDye^™ II and small LDs were imaged by confocal microscopy (Ex 473 nm/ Em 490-540 nm) and STED microscopy (Ex 473 nm/ Em 500-640 nm, depletion laser 660 nm). STED imaging detected ~120 nm (FWHM) small LD, which was not clearly detected by confocal microscopy. Detailed STED imaging condition and analysis methods were described in original paper below.

Observation of microglia using two-photon microscopy
LipiDye^™ II (1 uM) was added to primary cultured rat microglia, stained overnight, fixed with 4% PFA, and observed with two-photon microscopy (excitation 800 nm / fluorescence 510-560 nm). LipiDye^™ II can also be excited by two-photon excitation method and LDs of various sizes in primary cultured microglia could be detected.


*Data courtesy of Dr. Hyun Beom Choi and Dr. Brian MacVicar, The University of British Columbia.

Fig. 1 Comparison data between LipiDye and Nile Red
Lipid droplet in Adipocyte is stained with LipiDye or Nile Red. Nile Red also stains cytoplasm, while LipiDye specifically stains lipid droplet clearly.
* General filter for green fluorescent (e.g. FITC or GFP, etc) is not compatible with LipiDye. Please use an appropriate filter.

Fig.2 Preadipocytes differentiation
Differentiation process of preadipocyte (adipose progenitor cell) : 3T3-L1 is observed with LipiDye. Along with differentiation, lipid droplet also becomes grows in size.

Fig.3 Application of LipiDye in both post-fixed and pre-fixed cells
LipiDye is applied to cultured adipocytes after (left) or before (right) paraformaldehyde fixation. Regardless of fixation timing, LipiDye detects lipid droplet highly sensitive.

* Methanol fixation is not recommeded as it affect to the structure of lipid droplets. Please use cross-linking fixatives, such as paraformaldehyde.

Yamaguchi, et al., Angew. Chem. Int. Ed., 54:4539-4543 (2015).

Please down load the movie from here.