Sudan Red III in situ staining of cultured primary rat hepatocytes

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Scope of the method

The Method relates to

Human health

The Method is situated in

Basic Research
Translational - Applied Research

Type of 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.