Resource-directed foraging of the Neotropical mistletoe Struthanthus flexicaulis (Loranthaceae).

Some parasitic plants are capable of vegetative propagation, which allows them access to new hosts and improves nutrient availability. We aimed to determine what factors positively influence this propagation in the xylem-tapping Struthanthus flexicaulis, focusing on the use of directed foraging as a means of optimising access to resources. The study site was a rupicolous plant community in southeast Brazil. We evaluated how the success of branch propagation (by contagion) of the mistletoe is influenced by the crown height and density of its main host, the legume Mimosa calodendron. Oriented foraging was tested through comparing the response of mistletoe branches toward a potential host (resources) or a plastic net (no resources). Successful contagion was related to high density and the departure height of a branch from a host crown. In a high-density area, 89% of branches reached a new host versus 21% in a low-density area. Formation of an appressorium on either the plastic nets or hosts elicited branch growth reorientation towards it, as well as development of new branches. These responses were significantly stronger towards hosts than nets, and attributed to effective xylem tapping. The foraging strategy of S. flexicaulis benefits from branch response (growth and reorientation) to the development of appressoria and their resulting haustoria. The growth of appressoria on non-resource substrates and the ensuing response optimises the mistletoe spread by allowing these substrates to act as physical support for spreading branches and to infect a host that was previously out of reach.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Trypan blue exclusion assay by flow cytometry.

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.