Monolayer cell preparation

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

ATII cells, when plated on permeable supports or plastics under appropriate culture conditions, acquire features of type I cell-like phenotype and morphology [30, 57, 80], Although isolation of ATI pneumocytes from rat lungs has recently been reported with some success [28, 48, 81], development of confluent ATI cell monolayer with electrically tight characteristics has not been reported yet. It should be noted that unlike many other cells in primary culture, AEC exhibits generally a very limited proliferation profile and is therefore not suitable for passaging. Thus, a new preparation of cells has to be used for each data set, which drives the costs up tremendously, and a reliable normalisation scheme of data observed from each set of cell preparations is needed. 

Zhong and co-workers [530] described recent results of an investigation of the electrocatal3dic oxidation of methanol using carbon-supported An and Au-Pt nanoparticle catalysts. The exploration of the bimetallic composition on carbon black support was aimed at modifying the catalytic properties for the methanol oxidation reaction at the anode in direct methanol fuel cells (DMFCs). An and Au-Pt nanoparticles of 2-3 nm sizes encapsulated in an organic monolayer were prepared, assembled on carbon black materials and treated thermally. The results have revealed that these Au-Pt nanoparticles catalysts are potentially viable candidates for use in fuel cells under a number of conditions [530],... 

In vitro Agar diffusion test L-929 mammalian fibroblast cells Prepare monolayer cell culture with medium containing agar, place the flat surface of samples and controls in contact with the agar surface Size of the zone around or under sample... 

Tobey, R., Anderson, E., and Petersen, D. (1967). Properties of mitotic cells prepared by mechanically shaking monolayer cultures of Chinese hamster cells. J. Cell. Physiol. 70, 63-68. 

Fig. 1. Time course (A) and dose dependence (B) of NAD" " uptake by adult rat hepatocytes in primary monolayer culture (4). A, the hepatocyte monolayers were incubated in the presence of 500 iM NAD+ in the medium (5 ml/dish) and the intracellular NAD concentration was determined at the times indicated (4). B, hepatocyte monolayers were incubated with various concentrations of NAD" " in the medium, and the intracellular NAD+ concentrations were determined (4) after 60 min. The values are expressed as percent of untreated cells not incubated with NAD+. Control levels (= 100%) 1.3 0.082 nmol NAD+/mg protein (mean SEM, n = 8), which corresponds to an intracellular NAD+ concentration of 400 25 iM (mean SEM, n = 8). The values represent , single determination,, average of two determinations, mean SEM of 3 independent determinations, or mean SEM derived from 8 independent determinations, all involving separate cell preparations.

The relation between glucagon binding and stimulation of Na -depen-dent AIB uptake has been studied for isolated hepatocytes in suspension (25,50,67) or in monolayer culture (25). The dose-response curves for induction of transport were similar in both cell preparations despite the fact that the cultured cells contained more glucagon binding activity than did the freshly isolated hepatocytes (25). The half-maximal stimulation occurred at 0.5 to 1.5 nM glucagon (25, 62), whereas the maximal... 

Most of the characteristics for hepatic System A activation by insulin are remarkably similar to those already discussed for glucagon. Insulin-stimulated Na -dependent AIB transport has been demonstrated in freshly isolated cells in suspension as well as in monolayer cultures with only minor differences in the sensitivity between the two cell preparations (25). Like the glucagon induction of hepatic System A-mediated transport, the stimulation by insulin is preceded by a lag period of 30 - 60 minutes (54, 68), the degree of enhancement seen during a 2- to 4-hour incubation is only partially suppressed if the insulin in the medium is removed after the first 15 minutes (64,68), the effect is blocked by either cycloheximide (54,64,68) or antinomycin (68), the half-maximal stimulation of transport occurs at approximately 1 nM of hormone, and the... 

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

The ability to measure changes In an L-B film due to the presence of water vapor Is shown In fig. 7a-g and 8a-g. In this experiment the spectra of 2 monolayers of cadmium arachldate on N1 (tall to tall) are recorded In the presence of 11 torr of water vapor In nitrogen at 30 deg C and compared with the spectra obtained with dry nitrogen. The difference between cadmium arachldate on nickel and on silver Is expected to be small because both films are prepared with the same water bath L-B technique prior to transfer to the metal [16]. In both the hydrated and anhydrous experiments, the gas Is swept continuously through the cell to maintain constant pressure. Figures 7a-g show a sequence of dry and wet L-B film spectra In the C-H stretching region 3000 to 2800 cm-1. The spectra, a, c, e, and g of the anhydrous bllayer show the typical bands of fresh, unheated arachldate monolayers. 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

Plaques may be obtained for animal viruses by using animal cell-culture systems as hosts. A monolayer of cultured animals cells is prepared on a plate or flat bottle and the virus suspension overlayed. Plaques are revealed by zones of destruction of the animal cells. 

Fig. 6.2. Caco-2 epithelial cell monolayers cultured with T. spiralis L1 larvae in (A) the absence or (B) presence of 1 mg ml 1 rat monoclonal, tyvelose-specific antibody 9D4 (McVay etal., 2000). Monolayers were fixed and stained with trypan blue as described in ManWarren etal. (1997). (A) Serpentine trails of nuclei in dead cells are evident, revealing the paths travelled by larvae. (B) Tyvelose-specific antibody has inhibited the migration of the larva such that it is encumbered in cell debris and has pulled up a large area of the monolayer, creating a plaque (P). Bar = 50 urn. Photomicrograph prepared by C. McVay, TTUHSC, Lubbock, Texas. 

Fig. 6.3. Electron micrograph revealing the location of larvae in polarized Caco-2 monolayers grown on filter inserts (ManWarren etal., 1997). Apical microvilli provide evidence of epithelial cell polarization. Epithelial cell cytoplasm is evident above and below the larva. Bar = 2 pm. The position of the filter substrate is marked (F). Photomicrograph prepared by S. Pearce-Kelling and J. Ailing, Cornell University. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

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