The Selection of Serum-independent PC12 Cells for a More-reliable Manganese Cytotoxicity Test

A major issue concerning the protocols of heavy metal cytotoxicity tests with PC12 cells was the hypothesis that serum in the culture medium might sequester the metal, thus altering the results obtained. However, serum withdrawal impairs the viability of PC12 cells themselves, thus impeding cytotoxicity testing in the absence of serum. In this study, we repeatedly selected undifferentiated, totally non-adherent PC12 cells in Petri dishes. Surprisingly, we discovered that these cells could survive and proliferate in serum-free medium. Moreover, features such as NGF-responsiveness, resazurin reduction potential, doubling rate, protein content, and basal caspase-3 enzyme activity, were equivalent to those exhibited by standard PC12 cultures. Further experiments aimed at fully characterising these serum-independent PC12 cells are in progress. These cells enabled cytotoxicity experiments to be conducted with manganese, both in serum-supplemented and in serum-deprived medium. The results demonstrated that serum removal decreased the LC50 of manganese from 250μM to 32μM, without affecting the internalisation of the metal. The data exclude an early competitive effect of serum on metal internalisation; rather, they suggest a late protective mechanism mediated by serum against the cytotoxic effect of the already-internalised metal.

Energy Transfer between Fluorescein Isothiocyanate and Propidium Iodide – A Problem in the Estimation of Tpotwith the Bromodeoxyuridine–DNA Flow Cytometry Technique?

Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 Å) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fullfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine – DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2–3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used.