Cultures, tissue samples

Quantification of the cellular response to irradiation was carried out using several biophysical parameters for in vitro-cultured cells and tissue samples ... 

Studies of the enzyme content of cells frequently involve the use of coarse tissue samples of either animal or plant origin. In such cases some preliminary dissection of the tissue may be necessary to isolate the relevant tissue components and remove unwanted structural material such as collagen, cellulose, etc., before moving on to the more critical disruption of the cells. Sometimes it is possible to use the technique of tissue culture to provide pure cell preparations for subsequent studies. 

Ethical issues as well as difficulty in obtaining enough human nasal tissue specimens have called for the need to use alternative in vitro and in vivo methods. Various in vivo animal models and in vitro excised tissue models have been described in the literature for nasal drug transport studies. However, due to the difficulty in both controlling the experimental conditions in in vivo animal models and obtaining intact excised tissue samples, in vitro cell culture models are also being actively developed. 

Despite the large amount of research done on these devices and the relatively detailed understanding of their operation, they have not yet found widespread use. There are several reasons. The main one seems to be the insufficient lifetime (both shelf and in-use ), due to the degradation of the ex vivo enzyme preparations. Particularly when they are used in real samples, various reversible and irreversible inhibitors limit their usable lifetime. This problem seems to be mitigated when the enzymes are used in their natural environment, in cell cultures, tissue slices, and even in whole organs (Arnold, 1986). 

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

In the same way, a 6-well culture plate has been prepared in order to monitor gradients in the oxygen supply of engineered tissue samples over a 21 day period. Figure 8 shows the horizontal cross-section of a tissue and the resulting charts of different samples after different time periods. light-scattering particles (TiCh) had been added to the polymer mixture to increase the excitation efficiency within the sensitive layer [33]. 

Laminar flow cabinets serve two purposes to protect the samples and to protect the worker and the environment. For most tissue culture applications sample protection is sufficient, but increasingly often we are becoming aware of hazards associated with biological samples and the cabinet to choose combines both aspects of protection. Such are the vertical laminar flow cabinets available, for example, from Flow Laboratories (Gelaire) or M.D.H. (Appendix 3). 

Cell culture systems are ideal for detailed proteomic investigations of responses in protein expression to controlled stimuli. This is because they should provide defined systems with much lower inherent variability between samples, particularly if established cell lines are used. However, cells that are maintained in culture respond by alterations in their gene pattern, and consequently the protein expression, such that it can be quite different from that found in vivo. This process can occur quite rapidly in primary cultures of cells established from tissue samples and is even more profound in cells maintained long-term, particularly where transformation has been used to establish immortal cell lines. Cardiac myocytes can pose an even bigger challenge. While neonatal cardiac myocytes can be maintained and grown in vitro, adult cells are terminally differentiated and can be maintained for relatively short times in vitro but are not capable of cell division. 

Immunobistocbemistry (IHC) is a generic term that describes tecbniques used to determine tbe presence of one or more antigenic proteins in cells and/or tissues. Samples suitable for immunobistocbemical analysis include smears and swabs, cells either cultured or in suspension, and tbin sections of body tissues. 

The traditional way to determine OXPHOS enzyme activities is by spectrophotometry. Several assays have been described for all 5 OXPHOS complexes. The assays are performed in homogenates of tissue samples or cultured cells, in crude mitochondria-enriched 600 g supernatants of tissue/cell homogenates, or in mitochondrial preparations from 14000 g pellets derived from 600 g supernatants. Obviously, the higher the... 

Janssen AJ, Trijbels FJ, Sengers RC, Smeitink JA, van den Heuvel LP, Wintjes LT, Stoltenborg-Hogenkamp BJ, Rodenburg RJ. Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. CUn. Chem. 2007 53 729-734. 

Quantitation and standardization of the amounts of total DNA isolated from each tissue sample must also be performed prior to analysis by Southern blot hybridization so that the amounts of total DNA loaded into each lane are equivalent and so that the autoradiographic signals obtained after Southern blot hybridization are directly comparable. This is not required for the analysis of DNA isolated from tissue culture as the number of PDH extracted are standardized at the time of seeding. By recording the exact weight of the tissue extracted, it is possible to calculate the viral load per cell in each sample. 

The diagnosis of HP infection can be made using endoscopic or nonendoscopic tests (Table 33-6). The tests that require upper endoscopy are more expensive, uncomfortable, and require a mucosal biopsy for histology, culture, or detection of urease activity. Recommendations to maximize the diagnostic yield include taking at least three tissue samples from specific areas of the stomach, as patchy distribution of HP infection can lead to false-negative results. Because certain medications may decrease the sensitivity of these tests, antibiotics and bismuth salts should be withheld for 4 weeks and PPIs for 1 to 2 weeks prior to endoscopic testing. 

Samples for this assay may be cell supernatant from cell culture experiments, cell lysates, tissue lysates, or biological fluids such as serum or plasma. High degree of hemolysis in serum or blood in tissue samples interferes with the measurement due to the presence of erythrocyte acid phosphatase. 

Fluorescence in situ hybridization (FISH) is a common technique used to detect gene rearrangements, regions of chromosome deletion and amplification, and numerical chromosomal abnormalities. It utilizes fluorescently labeled DNA probes that bind to homologous chromosomal regions and can assay interphase nuclei. FISH can be performed on a variety of tissue specimens including frozen tissue or FFPE tissue sections, touch preparations from fresh or frozen tissue samples, cultured cells, and cytologic smears. 

Implicit in the discussion of the chemistry of formation for DNA adducts in vitro is the generation of DNA adducts in vivo. The previously described pyrimidopuri-none, propano, and etheno adducts are found in human tissue samples and cultured human cell lines. The estimated levels of these adducts range from one to 10 adducts/108 bp DNA. Owing to the relatively low abundance of these lesions amid a preponderance of impurities, highly sensitive and specific analytical... 

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