Intracellular flow cytometric lipid analysis – a multiparametric system to assess distinct lipid classes in live cells

Abstract

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content: intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric SPADE analysis of hepatic cellular lipid distribution including Vitamin A autofluorescence is presented. This flow cytometric system allows for the rapid, inexpensive, non-destructive identification of lipid content and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Investigating the role of lipid genes in liver disease using fatty liver models of alcohol and high fat in zebrafish (Danio rerio)

“…The copyright holder for this preprint this version posted April 14, 2023. ; https://doi.org/10.1101/2023.04.14.536511 doi: bioRxiv preprint accumulated in our models. [29,32] Knockdown of our candidate genes specifically increased hepatic neutral lipids compared to free fatty acids in the presence of both EtOH and HFD, suggesting that these three genes regulate triglyceride metabolism in ALD and NAFLD models.…”

“…A set of 5-8 zebrafish larvae were stained in E3 solution (300 µL) containing Nile Red (300 nM) and Nile Blue (5 µM) for 30 minutes [29]. Zebrafish larvae were anesthetized using tricaine.…”

Investigating the role of lipid genes in liver disease using fatty liver models of alcohol and high fat in zebrafish (Danio rerio)

“…containing Nile Red (300 nM) and Nile Blue (5 μM) for 30 minutes. 29 Zebrafish larvae were anaesthetised using tricaine. For imaging, larvae were immobilised using 1% w/v low melting point (LMP) agarose dissolved in E3 solution.…”

New boron-based coumarin fluorophores for bioimaging applications

“…Co-localisation studies were first conducted against Nile Red, which marks lipid droplets in A549 cells. [20,21] Lipid droplet homeostasis is critical for cell signalling and the production of inflammatory mediators, with lipid droplet accumulation associated in cancer, cardiovascular diseases and insulinresistance. [22] Co-staining studies of the highly lipophilic ortho-1,2-carborane derivatives (HCCb and ICCb) with Nile red revealed clear co-localisation (Fig.…”