MTT Trouble (viability assay)

Burton Maggi Burton at
Mon Mar 6 04:38:46 EST 2000

    I use MTT assay in order to measure  keratinocytes (HaCaT cells)
survival after UVB irradiation. As you probably know, this assay is
based on the reduction of the tetrazolium salt MTT by viable cells (dead
cells are unable to metabolize this salt). The reaction produces a
water-insoluble formozan salt inside the cells which is solubilized in
DMSO before reading the plate at 540nm.
    We usually observe 60% of survival after irradiation of the cells.
But since a while,  instead of observing a water-insoluble formozan salt
inside the surviving cells (visualized by blue points inside the cells),
we observe a water-insoluble formozan salt (visualized by blue crystals)
outside the cells! No blue crystals appeared in the incubation medium
without the cells. Due to this problem, we can't observe any more the
toxicity of UVB as we obtain the same results
for the control-cells and the UVB-irradiated cells.
Maggi Burton
University of Louvain-la-Neuve

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