The present standard procedure describes a protocol for measuring the urea concentration in supernatant of human stem cell-derived hepatocyte-like cells. This procedure relies on a chromogenic reagent that forms a colored complex specifically with urea. The latter can be measured and is directly proportional to the urea concentration in the sample.

Last updated on: 22-03-2019 - 11:30

Contact: Cannot be disclosed
Organisation: Vrije Universiteit Brussel
Partners: Cannot be disclosed
Status: History of useInternally validated

The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division.

Last updated on: 20-03-2019 - 17:26

Organisation: Sciensano
Status: Published in peer reviewed journalValidated by an external party (e.g. OECD, EURL ECVAM,…)

The Vitotox test is a high-throughput bacterial genotoxicity test. The test is based on bacteria that contain the lux operon of Vibrio fischeri under transcriptional control of the mutated recN promoter, which is controled by the bacterial SOS-system (TA 104-recN2-4 strain or Genox strain) . After incubation of the cells with a genotoxic product, the recN promoter will become derepressed, resulting in expression of the lux operon.

Last updated on: 20-03-2019 - 17:34

Contact: Roel Anthonissen
Organisation: Sciensano
Status: History of use

The method detects two facets of drug-induced cytotoxicity i.e. the intracellular accumulation of phospholipids and of neutral lipids, i.e. phospholipidosis and steatosis respectively. The assay makes use of a kit containing an aqueous, red-fluorescent formulation of labelled phospholipids (LipidTOX™ Red phospholipid stain, excitation/emission ~595/615 nm) which is up taken by the cells upon incubation with a phospholipidosis-inducing compound.

Last updated on: 22-03-2019 - 11:29

Contact: Kaat Leroy
Organisation: Vrije Universiteit Brussel
Status: History of use

This method assesses general cytotoxicity. Upon disruption of the cell membrane, lactate dehydrogenase (LDH) is released. LDH catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of reduced (NADH) and oxidized (NAD+) nicotinamide adenine dinucleotide. The principle of the assay described in the current standard operating procedure is based on this reaction.

Last updated on: 22-03-2019 - 11:28

Contact: Kaat Leroy
Organisation: Vrije Universiteit Brussel
Status: History of use

The RT-qPCR assay is used to identify genotoxic and non-genotoxic compounds. Herefore metabolic-competent human HepaRG cells are exposed to the IC10 value (measured by the MTT test). A microassay is performed to select 84 genes that show the most robust rates of correct classification of the reference compounds into the genotoxic or non-genotoxic group. Each qPCR array also consists of 5 housekeeping genes.

Last updated on: 20-03-2019 - 17:22

Organisation: Sciensano
Partners: Vrije Universiteit Brussel
Status: Internally validatedPublished in peer reviewed journal

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation.

Last updated on: 22-03-2019 - 11:27

Contact: Robim Rodrigues
Organisation: Vrije Universiteit Brussel
Status: Published in peer reviewed journalValidated by an external party (e.g. OECD, EURL ECVAM,…)

Human embryonic kidney (HEK) 293 FT cells is a celline that is very easy to culture and is used to obtain high viral titers. “293” is a reference to the 293th experiment wherein the cell line was discovered. A transfection with an adenovirus type 5 DNA fragment took place, causing the cell line to express E1A adenoviral gene. This stimulates the transcription of specific viral genes, resulting in a high production of viral proteins.

Last updated on: 22-03-2019 - 11:28

Contact: Matthias Rombaut
Organisation: Vrije Universiteit Brussel
Status: History of use

The neutral red uptake (NRU) assay provides a quantitative measurement of the number of viable cells. The test is based on the ability of living cells to take up and bind neutral red (NR), a dye which easily penetrates cell membranes via non-ionic diffusion. It accumulates in the lysosomes. Dying cells have altered membrane properties and therefore they cannot take up neutral red (NR) anymore.

Last updated on: 20-03-2019 - 17:11

Contact: Roel Anthonissen
Organisation: Sciensano
Status: History of use

The Alkaline Comet Assay is a microgel electrophoresis technique which allows to measure DNA damage (single and double strand breaks, alkali labile sites, incomplete excision repair sites and cross links) cell by cell. Cells are mixed with 0.8% Low Melting Point Agarose which is spread as a gel onto a microscope slide. The cells are lysed with high salt concentrations and detergents.

Last updated on: 20-03-2019 - 17:30

Contact: Roel Anthonissen
Organisation: Sciensano
Status: History of use