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. 2000 Jan;32(1):63–75. doi: 10.1023/A:1008121125755

Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities

Gale Haslam 1, Diane Wyatt 1, Paul A Kitos 2,✉
PMCID: PMC3449446  PMID: 19002967

Abstract

A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD+, diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(ΔA490 = A490, test – A490, control) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (– Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors.

Keywords: indirect cell count, lactate dehydrogenase, LDH, tetrazolium reduction, toxicity assay

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