Cell viability measuring

Fig. 15. Effects of turbulent shear stress level and exposure time on cell viability measured by trypan blue staining. Cells were sheared in a concentric cylinder viscometer [1]...

PMs based on the EPISKIN in vitro end point were developed from the data obtained during the ECVAM Skin Corrosivity Validation Study (Barratt et al 1998 Fentem et al., 1998). The EPISKIN data are cell viabilities, measured following treatment for 3 minutes, 1 hour, and 4 hours. 

Analogous to cell viability measurements, many of the same basic concepts apply to the development of cell-based assays for apoptosis. The length of incubation of cells with the test compound is among the most important issues to address and optimize. The length of incubation is important because the markers of apoptosis may be present for relatively brief transient periods and subsequently disappear as the population of cells undergoes secondary necrosis. The induction of measurable caspase activity can occur in only a few minutes or can take days, depending on the model cell line, type of inducer, and effective concentration inside the cells. 

The EpiOcular model uses normal human-derived epidermal keratinocytes, which form a stratified squamous epithelium morphologically similar to that found in the cornea [36]. The main endpoint evaluated is cell viability measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Thiazolyl blue EINECS number 206-069-5, CAS number 298-93-1), into a blue formazan salt that is quantitatively measured after extraction from tissues. Two different protocols exist, the EpiOcular ET50 mainly developed for the testing of surfactants and surfactant-based mixtures, and the EpiOcular Eye Irritation Test (EIT) developed for a variety of chemistries. 

The cell viability measured by MTT assay is a very sensitive colorimetric assay, and only viable cells with intact mitochondria can reduce the dye tetrazolium (MTT). There is a direct proportionality between the formazan produced (expressed as Optical Density) and the number of viable cells." ... 

Figure 16.13. (top) Marrow stromal cell adhesion and proliferation on polystyrene, titanium and nanotubular surfaces for up to seven days of culture, nanotubular surfaces show approx. 40% more cell proliferation after seven days of culture compared to titanium surface (p < 0.05) (bottom) Cell viability measured as absorbance using MTT assay after four days for cell culture on polystyrene, titanium and nanotubular surface. 

Fig. 1 Results of cell viability measurement accoding to culture term. Cell proliferation amount was represented by optical density (O.D) (a) Measured O.D values of MCF-7. Measured O.D values of MG-63...

Models based on Eqs. (47)-(50) have been used in the past to describe the disruption of unicellular micro-organisms and mammalian (hybridoma) cells [62]. The extent of cell disruption was measured in terms of loss of cell viability and was found to be dependent on both the level of stress (deformation) and the time of exposure (Fig. 25). All of the experiments were carried out in a cone and plate viscometer under laminar flow conditions by adding dextran to the solution. A critical condition for the rupture of the walls was defined in terms of shear deformation given by Eq. (44). Using micromanipulation techniques data were provided for the critical forces necessary to burst the cells (see Fig. 4)... 

A dual isotope labeling technique [85] has been used to measure membrane permeability in plant cells, based on the selective permeabiHty of the membranes of living cells to tritiated water and carbon-14 labeled mannitol. Kieran [29] showed that the results of the dual isotope labeling and Evan s Blue staining methods correlated well as indicators of cell viability however, the latter was preferable in terms of reagent cost and ease of analysis. 

ATP is an ideal indicator of cell viability. Blood or blood cell concentrates prepared for transfusion are stored for periods of a few days to several weeks in the blood bank. Viability checking of the blood cells is necessary to avoid posttransfusional reactions [94], This quality control of the conserved red blood cells and platelets can easily be performed by measuring the ATP concentration as an expression of their integrity. By the same measurement it was possible to confirm the diagnosis and monitor the treatment effects in various cases of platelet disease [97], The possibility of determining cells viability can be exploited to examine more free cells or tissue, as in the spermatozoa viability test, based on the correlation between ATP content and mobility. 

From Table 4 it is evident that several natural and modified polyanions did not induce insulinoma cell detachment. Furthermore, the DNA content, a quantitative metric of cell viability, was in general near the baseline (100%) observed in the control studies with PBS. In generalboth the cell attachment and DNA indices gave similar indications of cytotoxicity. Therefore, in an effort to unify these measures the third column of Table 4 reports an overall ranking of the polymer... 

Low levels of mercuric chloride in polymorphonuclear cells may profoundly alter the cell respiratory burst, measured as chemiluminiscence, oxygen consumption and H2Oz production [171-173], depress phagocytic capacity [172, 173] and enhance release of lysosomal enzymes [ 172] with minimal loss of cell viability. A stimulation of oxygen metabolism in vivo might promote tissue injury, via the local production of free oxygen metabolites, in addition to depression of host defence [173],... 

The human skin model assay involves measuring the effects of corrosives on viable cells in a reconstituted human skin equivalent. To be accepted as a valid human skin model, several criteria must be met. The artificial skin must comprise a functional stratum corneum with an underlying layer of viable cells. Furthermore, the barrier function of the stratum corneum, as well as the viability of the epidermis, must be verified with appropriate experimental setups. The chemicals to be tested are applied up to 4 h as a liquid or a wet powder onto the skin model. Afterwards, careful washing has to be performed, followed by investigation of the cell viability [e.g., with a (MTT)] reduction assay). 

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number <100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

The supernatant was replaced after 3 days of exposure of cells (tumor and normal) to plant extracts and the cells were washed with PBS, then 500 pL MTT solution (0.25 mg/mL) was added to each well. The cells were washed after 3 h of incubation at 37 C, the formazan crystals formed in hving cells solubilized with 1 mL isopropanol, and the absorbance measured at 570 run with a Jasco UV-Vis spectrometer. Cell viability was expressed as a percentage of control treated with different concentrations of plant extracts. 

XTT assay is suitable for measuring cell proliferation, cell viability or cytotoxicity. The tetrazolium salts are converted into a coloured formazan product by cellular enzymes present in the mitochondria of a metabolically active cell. These enzymes are rapidly inactivated when a cell dies, and hence the activity of these enzymes can be used to monitor the viability of a cell. 

It has been shown that radio frequency impedance (RFI) is an effective tool for moifitoring cell density and cell growth of bioprocesses. The fermentation process, quite complex, is oftentimes difficult to sample and monitor. The RFI measurement could detect cell viability of Escherichia coli during the fermentation, serving as a qualitative measure of the metabolic load of the cell, and thus provide an in situ indicator of the optimal harvesting times. 

Treatment of cells with concentrations of vincristine or vindesine that produce relatively little effect on cell viability results in an accumulation of cells in the M and Gj (gap after DNA synthesis) phases of the cell cycle. The cellular effects, as measured by techniques such as flow... 

The hrefly Inciferin system is very sensitive and can be conpled to any enzymatic reaction that prodnces or nses ATP. For example, creatine phosphokinase can be determined by this method and hence be nsed in the diagnosis of myocardial infarction and mnscle disorders. The creatine phosphokinase converts AMP into ATP which then nndergoes the reaction with Inciferin as shown in Fignre 3.25. ATP pro-dnction is essential for every known life form and the firefly Inciferin system can be nsed to check for microbial life. Hence systems have been developed that use a portable luminescence workstation to monitor sanitation in food manufacturing and to check for sterile environments in technological workplaces. The system can also be applied in checking cell viability, for instance in cell cultures and to measure the toxic effects of chemicals on cells. 

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