Analysis of mammalian viable cell biomass based on cellular ATP

Abstract

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

“…This value represents the mean and standard deviation from triplicate experiments. This is in good agreement with the reported value of 2 – 6 fmol ATP per viable mammalian cell [44]. With E. coli , the relationships between cell number and both bioluminescence (Fig.…”

A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

“…As cell number was at maximum from cells grown from an inoculum of 2000 cells/mL, this signal decrease could be a function of cells reaching a stationary phase of growth and therefore the ATP production decreasing. This phase related decline in ATP production has been observed in other cell lines [29-31] and is synonymous with a high cell density in fibroblast cell culture [32]. Cell density has been shown to regulate trypanosome number in infections by differentiation induced by stumpy induction factor at high concentrations [33], and it could be suggested that other metabolites may be produced at higher cell densities which effect cultures in monomorphic forms.…”

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“…Hemocytometer counts in this range of perfusion rates were close to the VCM readings. However, below 0.2 nL cell −1 day −1 , the viability of the culture declined, the VCM readings underestimated hemocytometer counts by approximately 35%, though this may be due to decreasing cell volumes under low viability conditions (Sonderhoff et al, 1992;Ducommun et al, 2002). These results contrast with Wu et al (1995) who reported that several optical probe outputs overestimated viable cell concentrations (since nonviable cells scatter light).…”