Permeable Cell Assay

Permeable Cell Assay A Method for High-Throughput Measurement of Cellular ATP Synthetic Activity... 

Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity (4). In this assay, osmotic shock and Triton X-100 treatment made bacterial cells permeable for ATP Discharged ATP reacted with external luciferase and is detected as bioluminescence. An increased bioluminescence is observed with permeable cells, whereas it is not observed with standard ATP solution and heat-inactivated permeable cells. The cellular ATP synthetic activity is calculated from the slope of increasing bioluminescence. Permeable Cell Assay is simple and rapid with a small amount of cell culmre for quantification of ATP synthesis. 

The Permeable Cell Assay was optimized for E. coli K-12 (BW25113) (4). To investigate applications of this assay, P. fluorcscms, P. putida, C. ammoniagmcs, and C. glutumicum were tested. All bacteria were grown in LBG medium at 30°C for 24 h. The final OD of each bacterium was adjusted to 0.1, and the intensity of luminescence was measured. The intensity of luminescence from all bacteria tested with various concentrations of Triton X-100 showed saturated luminescence with 0.4% Triton X-100 (4). Both species of Corynehacterium... 

The Permeable Cell Assay is a simple method to measure the cellular ATP synthetic activity. ATP should be synthesized from domestic ADP and externally added inorganic phosphate mainly by the glycolytic pathway with consumption of glucose. High concentration of inorganic phosphate inhibits luciferase. The used concentration of inorganic phosphate (1.5 mM) was determined that it does not inhibit the activity of luciferase too much. 

The interaction of adenyl nucleotides with the process of ADP-ribosylation was evaluated using a permeable cell assay system to allow cellular uptake of the substrate NAD. The system used was one in which the MCF-7 human breast cancer cells were... 

We then used this Caco-2 cell assay to categorize representative fluoroquinolone drug substance permeability [50], The drugs demonstrated some concentration-dependent permeability indicative of active drug transport. Based upon comparison to labetalol, ciprofloxacin was classified as a LP drug, whereas levofloxacin, lomefloxacin, and ofloxacin were classified as HP drugs, which matched their human in vivo bioavailabilities. All four fluoroquinolone drugs were subject to efflux transport (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). 

Preliminary pharmacokinetic behavior can be tested through a number of whole cell assays. Most commercially successful drugs are administered orally, meaning the drug must be able to enter the bloodstream by crossing membranes in the intestines. The most common membrane permeability assay is performed by monitoring the absorption and secretion of a compound by colon carcinoma cells (Caco-2). Diffusion across Caco-2 cell membranes is considered to be a valid model for molecular transport in the small intestines.16... 

PAMPA-biomimetic-Caco-2-comparison Several in vitro assays have been developed to evaluate the Gl absorption of compounds. Our aim was to compare three of these methods (/) the BAMPA method, which offers a HT, noncellular approach to the measurement of passive transport ( ) the traditional Caco-2 cell assay, the use of which as a HT tool is limited by the long cell differentiation time (21 days) and (// ) The BioCoat HTS Caco-2 assay system, which reduces Caco-2 cell differentiation to three days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (f= 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 assay system does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21 -day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption. 

For compounds where PAMPA showed much lower permeability than the Caco-2 cell assay, it can be concluded that absorption transport is the primary permeability mechanism for these compounds. 

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