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 desription

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)
Belgium