Flow cytometry of cell proliferation through the incorporation of bromodeoxyuridine as an index of growth rate in the water flea, Daphnia magna (Crustacea, Cladocera)

Abstract

In this paper, we used a small crustacean as a model to develop a method for quantifying growth rates through the measurement of a cell proliferation marker. This was done in order to study the feasibility of this assay for estimating zooplankton production in the ocean. Flow cytometry immunodetection of bromodeoxyuridine (BrdU) was performed to detect and quantify the cycling nuclei of Daphnia magna. METHODS: A combination of mechanical dissociation and cell enucleation procedures proved to be the most convenient method for preparing nuclear suspensions from whole organisms. Up to three populations of nuclei with different ploidy were observed. The relative abundance of these nuclear populations changed with the size of the flea. RESULTS: The staining technique has been optimized. The time and concentration for the maximum detection of BrdU-labeled nuclei were 3 h at 300 microM BrdU. Whole organisms can be frozen (-20 degrees C) after incubation with no changes in the final results. The method was used in different physiological conditions under controlled food and temperature in order to test the inverse relationship between physiological rates and size of organisms at several developmental stages. The quantification of BrdU-labeled nuclei in 1-6 day-old larvae showed the highest labeling index, with a mean of 95 +/- 1% (n = 22). In contrast, young animals (0.8-1.2 mm) had 25 +/- 4% (n =16, P < 0.001) and adults (>1.4mm) had 14 +/- 3% (n = 4, P < 0.001). The results obtained show an expected tendency, suggesting that a direct relationship exists between the labeling index and the instantaneous growth rate. CONCLUSIONS: Certain features of our method, such as the short times required for labeling and the possibility of preserving the samples during field experiments and under different conditions (including natural concentrations and types of food), are advantageous to the study of processes governing energy fluxes in pelagic ecosystems.