Dye exclusion

Capillary shear tests were performed on low density (50 g fresh weight 1" ) suspensions of M. citrifolia using the apparatus illustrated in Fig. 1. Under both laminar and turbulent conditions [54,120,121], the relative viability of the suspension, evaluated using the Evan s Blue dye exclusion technique, was found to fall with exposure time in the loop. Loss of viability is well described by a first-order model ... 

When tested on cell counts, dye exclusion and fine structure of guinea-pig thymocytes, mercuric chloride in concentrations which inhibit DNA synthesis, most often caused cell degeneration, with condensed nuclear chromatin and disintegrating cytoplasmic organelles [170]. 

Figure 1. Inhibition of geranylgeranoic acid-induced apoptotic cell death by a-tocophrol. Increasing concentrations (10 100 pM) of a-tocopherol were added to HuH-7 cell cultures with (closed circle) or without (open circle) 10 pM geranylgeranoic acid. After overnight incubation, the number of the viable cells was counted using a Trypan blue dye exclusion method. Means S.E. (n=3) are shown.

The antioxidant activity afforded by NO is most important if it translates into a meaningful therapeutic event. To test the importance of this reaction pathway to cell survival, we compared viability of the cells exposed to in the presence or absence of NO, When cells are exposed to 20 pM there was appreciable loss of cell membrane integrity as measured by trypan blue dye exclusion (Kelley et al, 1999) the addition of NO deaeased cell membrane damage (Figure 6). 

Trypan blue dye exclusion, Lactate Dehydrogenase (LDH) release... 

This investigation aims for the first time at the stabilization of photoisomers of photochromic dye molecules in molecular sieves by modification of the MCM-41 surface and covalent bonding of dye molecules. Diphenyldichlorosilane is utilized for pre-silylation to warrant an anchoring of the dyes exclusively inside the MCM-41 channel structure. 

Cell concentration in suspension can be determined through an optical microscope employing a hemocytometer for manual cell counting, or in a semi-automatic way using an electronic particle counter (such as a Coulter counter), as described in detail by Freshney (2005). Through dye exclusion (such as trypan blue), it is possible to determine viable cell concentration, that is the number of cells in a known sample volume capable of proliferating in favorable culture conditions. 

Evaluation of membrane integrity is the most commonly used measurement of cell viability, indicating instantaneous or progressive damage over a few hours, and can be performed by 51Cr or specific enzymes release or a dye exclusion assay. These assays are particularly important for toxic agents that exert a primary effect on membrane integrity. Nevertheless, quantification can be difficult due to cell loss by surface detachment or autolysis. 

The methodology normally used for the determination of dead cell concentrations is the dye exclusion method (Freshney, 1994), although this is not adequate in cases in which cellular lysis is significant. In these situations, other methodologies must be adopted to determine the amount of lysed cells, such as, for example, the measure of the lactate dehydrogenase concentration (LDH) in the culture medium (Freshney, 1994 Miller and Reddy, 1998). 

Regrowth potential Membrane integrity Dye exclusion Dual isotope labelling Dielectric permitivity... 

The prepared cell line is collected on the 7th day is washed, resuspended in complete medium containing GM-CSF and IL-4 and 2 mL and is plated in the wells of a six-well culture plate. lmL of test chemical also prepared in complete medium with GM-CSF and IL-4 is added to each well and the plate is incubated for 24 h. Viability of the DC after test chemical exposure is then assessed by propidium iodide dye exclusion using a Coulter Epics XL flow cytometer (Ryan et al. 2004). 

Cells are harvested from the dish by EDTA treatment, resuspended and rinsed in PBS-CMF, and finally resuspended at the desired concentration in serum free medium buffered at pH 7.2 with Hepes and filtered through 0.22 fim MiUipore membranes. Cell viability is checked by trypan blue dye exclusion test, and aggregates, if present, can be removed by filtration through cell strainers (Costar). 

At the end of the hypoxic period, viability of the cells were assessed by the Trypan Blue dye exclusion criteria. Fig. 15 demonstrated that the viability of hypoxic cells treated with IL were not significantly different form the viability of the normoxically cultured H9C2 cardiocytes. The viability of hypoxic cardiocytes treated with plain liposomes or IgG-liposomes was significantly lower than either those of immunoliposome treated hypoxic cells or normoxica cells. Since there was no difference in viability in cardiocytes treated... 

The pH value of dialyzed solutions was within the 7.0-7.4 range that corresponds to the normal pH of biological systems. Taking into account that the average thickness of cell membranes is about 8-10 nm, we can suppose that the synthesized nanoparticles ( 2 nm) can penetrate through the cell membranes. The particles can be used in native, solubilized and modified forms. The senthisyzed nanoparticles were tested on the rat hepatocyte cells using the standard procedure of trypan blue dye exclusion [7]. For a particle concentration up to 50 mg/1, the toxic effect has not been detected. 

The release of LDH activity can be related to the total number of dead and lysed cells, as determined by conventional dye exclusion methods. This relationship allows the growth and death kinetics within the culture system to be evaluated. However, there are various phenomena that could complicate this evaluation (Marc et ah, 1991 Legrand et al, 1992 Wagner et al., 1992). 

It is assumed that the release of LDH occurs rapidly after damage to the cell membrane. However, this assumption is not necessarily correct. The release of LDH can be complete in cells that are considered dead by dye exclusion methods. Alternatively, complete release may occur only upon cell lysis. This point is further complicated because dye exclusion methods do not measure lysed cells. 

There are several assays available to quantitate cell death in bioreactors. The simplest assay uses Trypan blue dye exclusion from intact cells to determine hve and dead cell concentrations. Cells that have died and lysed, however, cannot be accounted for using this assay (see Chapter 2, section 2.2). 

Cell damage under different agitation conditions can be assessed by monitoring the increase in LDH release. To equate the LDH content in culture supernatant to the number of cells that have lysed, a standard curve must be established for each cell type and (where applicable) for different environmental conditions to which the cells were exposed. The procedures for the Trypan blue dye exclusion assay and the LDH assay are described in Chapter 2, sections 2.2 and 2.5. 

Dye exclusion Cellular damage by agents Chemical penetration Cell growth and development Cellular metabolism... 

Weisenthal, L. M., Dill, P. L., Kumick, N. B., and Lippman, M. E. (1983) Comparison of dye exclusion assays with a clonogenic assay in the determination of drug-induced cytotoxicity. Cancer Res. 43, 258-264. 

The alternative to dye exclusion as a measure of membrane integrity is the capability of the plasma membrane to retain a marker molecule. The marker can be the appearance in the medium of an intracellular molecule, such as an intracellular enzyme like lactate dehydrogenase (LDH). LDH release has been used for fish cell lines184, but experience with mammalian cells suggests that the use of LDH can be complicated by several factors162. 

Some information on environmentally relevant reversal agents is available from aquatic invertebrates. P-gp-related dye exclusion in the mussel M. califomianus was inhibited by the moderately hydrophobic pesticides pentachlorophenol, dacthal, chlorbenside and sulfallate, but not by DDT, DDD, DDE or the PCB Arochlor 125432. Similarly, Diesel-2 oil water-extracts increased the accumulation of vincristine in the gills of M. galloprovincialis91. In the freshwater mussel Dreissena polymorpha, water from the polluted Sava River increased the accumulation of fluorescent P-gp... 

Loss of cytoplasmic membrane function occurs early in necrosis and late in apoptosis (so-called apoptotic necrosis ). If, in a given experimental system, the mode of death has been adequately characterized morphologically and the assay timepoint judiciously chosen, loss of dye exclusion can serve as a useful, easy to quantify measure of cell death in either apoptotic or necrotic model systems. Commonly used dyes for this type of analysis include propidium iodide, 7-AAD, Sytox green, and ethidium homodimer. 

Cell viability was determined using trypan blue dye exclusion test. 

Assessment of the viability of the cells was performed after 24 h of hypoxia by Trypan-blue exclusion or by immediate further incubation of the cells with [3H]-thymidine (3HT). According to the trypan-blue dye exclusion data, almost all control HSs were nonviable. Plain liposomes provided some protection from hypoxic injury, probably, by nonspecifically sticking to cell surfaces and fortuitously sealing some of the cell membrane breaches. IPs completely prevented cell death, with a cell viability similar to that of normoxic cells. Hypoxic cells treated with nonspecific IgG liposomes demonstrated viability at the level of PL-treated cells. 

Cell viability. Exponentially growing HL-60 cells, 15 x 106 in 75.0 mL of RPMI 1640, were incubated with either ATRA, or UDMA or BDDMA for five days. The total cell number was determined every day using the trypan blue dye exclusion test. Both the cellular proliferation - as area under curves (AUC) - and cellular mortality were calculated. 

It is well documented that normal human bronchial epithelial cells release a variety of cytokines and chemokines, and proinflammatory cytokines such as IL-la, IL-1 3, and TNF-a stimulate their production in vitro (Fig. 11) [61], We cultured human normal and transformed bronchial epithelial cells, and studied the effect of EM, CAM, and RXM on IL-6 [17], IL-8 [60, 62] and GM-CSF [63] production. Among the antimicrobes tested, only 14-membered-ring macrolides EM, CAM, and RXM showed an inhibitory action on cytokine release by unstimulated and cytokine-stimulated human bronchial epithelial cells (Fig. 12). In contrast, a 16-membered ring macrolide josamycin (JM) failed to show such effects. LDH release assay, a trypan blue dye exclusion test, and a colorimetric MTT assay showed that this effect was not due to cytotoxicity. Percent inhibition of IL-8 protein release in human primary bronchial epithelial cells was 25.0 5.67% at lO M, which is comparable with clinically observed serum concentration. 

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