Sudan Red III in situ staining of cultured primary rat hepatocytes

Scope of the method

Alternative method relates to
  • Human health
Alternative method is situated in
  • Basic Research
  • Translational - Applied Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Rat
Type of cells/tissues/organs
Primary rat hepatocytes

Description

Method keywords
  • Formaldehyde fixation
  • Sudan Red III staining
  • Hematoxylin nuclear counterstain
  • Primary rat hepatocytes
  • Intracellular lipids
  • in vitro
Scientific area keywords
  • Toxicology
  • Hepatotoxicity
  • Steatosis
  • Drug-induced cytotoxicity
Method description

The standard operating procedure for Sudan Red III in situ staining of cultured rat hepatocytes describes how to detect one of the aspects of drug-induced cytotoxicity i.e. the intracellular accumulation of lipids or in other words steatosis, in primary rat hepatocyte cultures. It is based on the ability of a lysochrome, i.e. Sudan Red III diazo-dye to stain intracellular lipids. Additionally, subsequent application of hemalum, which is a complex formed by aluminium ions and oxidized haematoxylin, colours nuclei of the cells and thus enables their localisation. Red-coloured lipid droplets and blue nuclei are readily visible upon examination of the cells under a light microscope.

Lab equipment
  • Inverse-phase light microscope (Nikon Optiphot) ;
  • Oven (Thermo electron corporation, Heraeus, 60°C).

Pros, cons & Future potential

Advantages

The standard operating procedure for Sudan Red III in situ staining of cultured primary rat hepatocytes is easily applicable and allows a simultaneous screening of multiple compounds and/or multiple concentrations of the same compounds (to examine chemically induced steatosis).

Challenges

Sudan Red III stain has a high affinity to a broad range of lipids and consequently does not discriminate between e.g. neutral lipids and phospholipids. Therefore, it is of utmost importance to perform more than one assay or use a more specific assay.

References, associated documents and other information

Associated documents
Sudan Red III staining.doc

Contact person

Raf Van Campenhout

Organisations

Vrije Universiteit Brussel
Pharmaceutical and Pharmacological Sciences (FARM)
In Vitro Toxicology and Dermato-cosmetology
Belgium
Brussels Region