Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the con...Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift;a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.展开更多
Biofilms are recognised as an important contributor to bacterial resistance towards traditional antimicrobial treatments. Assessment of biofilm formation currently relies on a 96 well microtitre plate assay, which usu...Biofilms are recognised as an important contributor to bacterial resistance towards traditional antimicrobial treatments. Assessment of biofilm formation currently relies on a 96 well microtitre plate assay, which usually involves the colourimetric detection of stain (typically crystal violet) removed from previously stained biofilm. The amount of crystal violet released is then used as a quantitative indicator of the amount of biofilm formed. Currently, this is achieved by solubilisation of the stain by ethanol which results in partial decolourisation of the crystal violet stained biofilm which impacts the accuracy and reproducibility of this method. Herein, we describe a modified biofilm dissolving solution (MBDS) which produces a more uniform and reproducible colour release from stained biofilm through solubilisation of the biofilm architecture itself. Here we use crystal violet stained biofilms of P. aeruginosa strain PA0-1, to demonstrate an approximate two fold increase in crystal violet release by MBDS, as compared to ethanol treatment. In addition, when ethanol decolourised biofilms were treated again with MBDS, an almost equal amount of remnant crystal violet was recovered by dissolving the biofilm and the stain trapped within it. These results were reflected in microscopic analysis of ethanol treated and MBDS treated biofilm. Similar results were obtained when MBDS was used to decolourise and dissolve the biofilms of a number of other bacterial species highlighting the advantages of MDBS as a universal solvent for the colour detection of biofilm.展开更多
文摘Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift;a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.
文摘Biofilms are recognised as an important contributor to bacterial resistance towards traditional antimicrobial treatments. Assessment of biofilm formation currently relies on a 96 well microtitre plate assay, which usually involves the colourimetric detection of stain (typically crystal violet) removed from previously stained biofilm. The amount of crystal violet released is then used as a quantitative indicator of the amount of biofilm formed. Currently, this is achieved by solubilisation of the stain by ethanol which results in partial decolourisation of the crystal violet stained biofilm which impacts the accuracy and reproducibility of this method. Herein, we describe a modified biofilm dissolving solution (MBDS) which produces a more uniform and reproducible colour release from stained biofilm through solubilisation of the biofilm architecture itself. Here we use crystal violet stained biofilms of P. aeruginosa strain PA0-1, to demonstrate an approximate two fold increase in crystal violet release by MBDS, as compared to ethanol treatment. In addition, when ethanol decolourised biofilms were treated again with MBDS, an almost equal amount of remnant crystal violet was recovered by dissolving the biofilm and the stain trapped within it. These results were reflected in microscopic analysis of ethanol treated and MBDS treated biofilm. Similar results were obtained when MBDS was used to decolourise and dissolve the biofilms of a number of other bacterial species highlighting the advantages of MDBS as a universal solvent for the colour detection of biofilm.
