Monitoring cell proliferation in vitro with different cellular fluorescent dyes.

There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5th to 8th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Induced death of Escherichia coli encapsulated in a hollow fiber membrane as observed in vitro or after subcutaneous implantation.

The encapsulation of bacteria may be used to harness them for longer period of time in order to make them viable, while antibiotic treatment would result in controlled release of therapeutic molecules. Encapsulated bacteria Escherichia coli GFP (green fluorescent protein) (E. coli GFP) were used here as a model for therapeutic substance - GFP fragments release (model of bioactive substances). Our aim was to evaluate the performance of bacteria encapsulated in hollow fibers (HF) treated with antibiotic for induction of cell death. The polypropylene surface modified HF was applied for E. coli encapsulation. The encapsulated bacteria were treated with tetracycline in vitro or in vivo during subcutaneous implantation into mice. The HF content was evaluated in flow cytometer, to assess the bacteria cell membrane permeability changes induced by tetracycline treatment. It was observed that applied membranes prevent release of bacteria through the HF wall. The encapsulated in HF E. coli GFP culture in vitro proves the tetracycline impact on bacteria viability and allows recognition sequence of events within process of bacteria death. Treatment with tetracycline of the SCID mice for 8 hours proves the tetracycline impact on bacteria viability in vivo, rising the necrotic bacteria releasing GFP fragments. It was concluded, that the bacteria may be safely enclosed within the HF at site of implantation, and when the animal is treated with antibiotic bacteria may act as a local source of bioactive factor.