Evaluation of the cytotoxic effects of a compound on cell lines - Plateforme de chimie biologique intégrative de Strasbourg - PCBIS - UAR 3286 - Faculté de pharmacie

Introduction

The study of the cytotoxicity of a compound having a modulating activity on the response of the cells is essential.
It allows to verify that the active molecule does not induce any modification of the cell viability compared to untreated cells.
Several cell lines are available at PCBIS for the study of cytotoxicity.

Principle

Several cell viability assays are available at PCBIS.

1. MTT assay

The assay is based on the cleavage by mitochondrial succinate dehydrogenase of a yellow tetrazolium salt into blue formazan crystals.

The reaction is quantified by measuring the absorbance at 560 nm of the crystals solubilized in an organic solvent which is proportional to the number of living cells.

2. WST-1 assay

The assay is based on the cleavage by mitochondrial succinate dehydrogenase of a red tetrazolium salt into yellow formazan crystals. The reaction is quantified by measuring the absorbance at 450 nm of the soluble crystals in the culture medium which is proportional to the number of living cells.

Unlike MTT, this terazolium salt is cleaved by living cells into soluble formazan, so there is no subsequent solubilization step in an organic solvent.

3. CellTiterGlo Luminescent Assay (PROMEGA)

The test is based on the mono-oxygenation of luciferin to oxyluciferin by luciferase in the presence of Mg 2+ and ATP.

The reaction is quantified by measuring the luminescent signal which is proportional to the ATP present in the medium and thus to the metabolic activity of the cells.

4. Incucyte (SARTORIUS)

This apparatus allows to realize kinetics of cell proliferation in classical culture conditions under controlled atmosphere by quantifying cell confluence.

Protocol

Format	96 wells
Cellular models	Several cell lines available
Control molecule	Chlorpromazine
Incubation	48H à 37°C ; 5% CO₂ Possibility to adapt the incubation time to the biological model

Viability tests
	MTT	WST-1	CELL TITER GLO	INCUCYTE
INCUBATION	0.5 à 4 hours	0.5 à 4 hours	10 minutes	Continious
PARAMETER	MTS reductase	MTS reductase	ATP	Cellular morphology
SENSITIVITY	+	++	+++	+++
DETECTION	colorimetry 1 point	colorimetry 1 point	luminescence 1 point	cell confluence kinetic

Notes :

Prepare 50 µL at 10 mM or 3 mg of sample to test 5 concentrations (3 points/concentration)

Recommendation :

Ensure sample solubility in a solvent compatible with cell culture

Evaluation of the cytotoxicity of Chlorpromazine on HepG2 (ATCC HB-8065)

All models can be adapted to your request.