Flow-cytometric determination of neutral lipids

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
Human skin-derived adults stem cells

Description

Method keywords
  • Flow-cytometry
  • neutral lipids
  • quantitative
  • fluorimetric
  • in vitro
Scientific area keywords
  • Steatosis
  • stem cells
  • lipids
  • lipid accumulation
Method description

Using this method you can measure the (relative) lipid load in human skin-derived stem cells differentiated towards hepatic cells. This method could also be applied on other cell types (e.g. HepG2), since it is based on the following publication: "M. T. Donato et al., Chem. Biol. Interact. 181, 417–423 (2009)." Briefly: 1. Aspirate medium from the cell culture 2. Incubate 10’ with TrypLE (200 µL/well for 24- multiwell format) 3. Add 500 µL pre-warmed PBS (37°C) to every well and harvest the sample 4. Rinse with 500 µL PBS 5. Centrifigate according to the cell type 6. Resuspend in 1 mL PBS (containing BODIPY(TM) 1:2500 (see publication above)) on ice 7. 5’ before measuring + 1 µL Hoechst (+homogenize by pipetting) 8. Dilute 1:10 (PBS (4 °C)) before measuring to limit background signal 9. Measure signal (up to 100.000 events)

Lab equipment
  • Flow-cytometer ;
  • Cell culture equipment ;
  • Biosafety cabinet.
Method status
  • Internally validated

Pros, cons & Future potential

Advantages

Fast.

Challenges

Measuring many samples can be time-consuming.

Modifications

You can use also other cell types.

Future & Other applications
  • Drug testing (anti-steatotic drugs) ;
  • Assessing drug-induced liver steatosis (in vitro).

References, associated documents and other information

References

M. T. Donato et al., Chem. Biol. Interact. 181, 417–423 (2009)

R. M. Rodrigues et al., Stem Cells Dev. 23, 44–55 (2014)

Associated documents

Contact person

Joost Boeckmans

Organisations

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