Techniques to Monitor Cell Viability and Tissue Integrity

To assess the functional state of cells in in vitro models, a variety of assays is available. They are necessary either to ascertain the quality of the deployed cell culture or to quantify the damage induced by the fungal effector cells to the host target cells as a parameter of virulence during the infection experiment. Methods to determine cell viability and function reach from simple live/dead assays over assays that focus specifically on a molecular mechanism or a cell type specific function to the most complex molecular fingerprinting to record biochemical alterations.

Commonly used techniques in in vitro infections are end-point live/dead assays that are based on assessment of cell membrane integrity.

The most conventional one is the trypan blue (TB) exclusion test which can be visualized by light microscopy. However, TB exclusion may lead to overestimation of cell viability (Mascotti et al., 2000). In the chromium (51Cr) release assay target cells are labelled with Na251CrO4. The compound is taken up by the cells and retained for some time in the cytoplasm. If the target cell is damaged upon co-incubation with effector cells, gamma ray radiation generated during 51Cr decay can be measured in the medium. This assay was for example adopted to determine endothelial cell damage by C. albicans and C. neoformans (Ibrahim et al., 1993; Ibrahim et al., 1995). Here, EC monolayers were incubated with Na251CrO4, the unincorporated tracer was removed and EC were then infected with the pathogen. By including an uninfected control, the spontaneous 51Cr release by EC and the specific endothelial release of chromium due to cell injury can be precisely calculated. The system can be further refined to determine whether EC damage requires direct contact with the pathogen or is due to secreted compounds by inserting a membrane between effector and target cells.

To avoid the inconvenience which accompanies isotopic methods, two main types of non-radioactive assays, fluorescent techniques and enzymatic procedures, are available.

Viability tests by double staining with the DNA intercalating compounds acrid-ine orange and ethidium bromide (AO/EB) or propidium iodide (AO/PI) are based on the fact that AO is taken up by both viable and nonviable cells while EB and PI only can pass through disintegrated cell membranes. Thus, viable cells appear green under darkfield fluorescence microscopy while dead cells are red (Baskic et al., 2006; Mascotti et al., 2000). In non-adherent in vitro models, as with certain immune cell system (see below), the fluorescence assays can be read by flow cytometry. There are commercially available products that are based on this principle of differential fluorescence as the LIVE/DEAD viability kit (Invitrogen, Molecular Probes, Carlsbad; CA, USA).

Enzymatic tests can detect the extracellular presence of intracellular proteins as indicator of cell membrane damage. Activities of the lysosomal N-acetyl-beta-D-glycosaminidase (NAG) or the lactate-dehydrogenase (LDH) are measured for this purpose (Korzeniewski & Callewaert, 1983; Niu et al., 2001; Schaller et al., 2004). The metabolic activity of cells can be quantified by 3H-glucose uptake (Steele et al., 2000) or addressed by substrates that emit fluorescence after cleavage by intracellular esterases (e.g. CellTracker Green or calcein blue AM; both Invitrogen, Molecular Probes).

Widely used colorimetric tests based on enzyme function utilize the tetrazolium dyes MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide) and XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) (Meshulam et al., 1995; Mosmann, 1983). Both colourless salts are reduced by cellular functions to brightly coloured formazan products (for a review see Berridge et al., 2005). Thus, they also indicate metabolic activity as parameter for cell viability. While MTT forms an insoluble formazan and is therefore applicable in end-point viability tests, XTT converts into a soluble product which allows for real time assessment of viability. Both assays can be performed quantitatively in an ELISA reader format, but the crystals built by MTT must be dissolved (Meshulam et al., 1995). The exact enzymatic activities responsible for MTT and XTT reduction as well as their cellular localization are not well known, which might lead to erroneous results under certain circumstances (Berridge et al., 2005; Knight & Dancis, 2006).

A principle difference between the chromium release assay and the non-radioactive enzymatic tests has to be taken into account for the experimental design. The former permits a specific label of target cells, while the enzymatic activities measured in the latter might be present in both the target and the effector cells. Thus, appropriate controls have to be included that enable to correct for the contribution of effector cells to the assay.

Cell layer and tissue in vitro models require techniques that monitor their integrity specifically. A characteristic physical property of epithelia and endothelia is a high trans-epithelial/endothelial electrical resistance (TEER) established by tight junctions between the cells (Santaguida et al., 2006). The TEER has to be measured to confirm the validity of the barrier and to detect changes induced by infection with pathogens (Jong et al., 2001). The resistance values can be determined by electronic devices in real time and are expressed in ohm per square centimeter of the layer surface (Grainger et al., 2006; Neuhaus et al., 2006b). Additionally, the tightness of the layer can also be addressed by determining the permeability of indicative molecules as 3H-inulin, 3H-sucrose, Evans blue, or fluorochrome-conjugated dextrans (Jong et al., 2001; Neuhaus et al., 2006a; Raimondi et al., 2006).

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

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