Cell Sorting, Metabolism & Molecular Biology

Managing PI: Peter Carmeliet, Max Mazzone

Cellular Biology and Cell sorting (Max Mazzone, Peter Carmeliet)

Cell isolation and culture: A routine schedule for isolation of primary human venous endothelial cells from umbilical cords and primary pericytes from adipose tissue on a daily or weekly basis is operational in close interaction with the University Hospital Gasthuisberg (department of Gynaecology) and Heilig Hart ziekenhuis (departments of Gynaecology and of Plastic and reconstructive surgery), Leuven. Isolation of murine primary endothelial cells from different organs based on magnetic bead sorting or fluorescence assisted cell sorting (FACS) is operational as well.
Cell sorting: Flow cytometry for cell cycle analysis or for protein expression measurements, as well as cell sorting from in vitro cultures or from homogenized tissues (e.g. human, mouse and zebrafish endothelial cells from various tissue biopts) is done using a BD FACSVerse flow cytometer and a state-of-the-art high-speed BD FACS AriaIII cell sorter. The FACS AriaIII system is equipped with 4 lasers providing 16-color analysis capability, an ACD unit for sorting in plates and a sample collection cooling system.

Flow cytometry

 

Cellular Metabolism (Peter Carmeliet)

The XF24 Extracellular Flux analyzer from Seahorse Bioscience Inc. is used to measure the rate of change of dissolved oxygen and pH in the media immediately surrounding living cells cultured in a microplate. It is routinely used for analysis of mitochondrial respiration and glycolysis in the framework of the various ongoing cell metabolism studies.

XF24 seahorseseahorse result

         

In addition, metabolite level analysis (measuring up to 150-300 metabolites involved in diverse pathways) and 13C metabolic tracer studies are performed using gas and liquid chromatography based mass spectrometry in collaboration with the Metabolomic Expertise Center - Bart Ghesquière.

Molecular biology and Biochemistry (Peter Carmeliet)

All standard molecular biology and biochemistry technologies are operational. For gene silencing or overexpression in vitro and in vivo, a routine system is operational for the construction of lentiviral vectors and generation of lentivirus encoding shRNA or cDNA (wild type or mutant variants) combined with a fluorescent reporter as required.


References:

  • The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts. Kuchnio A, Moens S, Bruning U et al. & Carmeliet P. Cell Rep. 2015 Aug 11;12(6):992-1005
  • Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Schoors S, Bruning U, Missiaen R, Queiroz KC, et al. & Carmeliet P. Nature. 2015 Apr 9;520(7546):192-7.
  • Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Schoors S, De Bock K, Cantelmo AR et al. & Carmeliet P. Cell Metab. 2014 Jan 7;19(1):37-48