Tissue culture assay

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

Van Langendonck, N., Bottreau, E., Bailly, S., Tabouret, M., Marly, J., Pardon, P., and Verge, P. (1998). Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains. /. Appl. Microbiol. 85, 337-346. 

Clostridium difficile can be cultured from the stool, and toxins A and B can be assessed by different techniques (116). The most accurate method is still a cytotoxin tissue culture assay. This detects the cytopathic effect of cytotoxin B, which can be neutralized by Clostridium sordellii antitoxin, but it takes 24 8 hours to show a result. Alternative tests that produce faster results have been developed. A latex agglutination test lacks sensitivity and specificity, and does not distinguish toxigenic from non-toxigenic strains. An enzyme immunoassay for toxin A may be an acceptable alternative to the cell cytotoxin assay and the results are rapidly available. A dot immunobinding assay has not yet been extensively studied (164). 

Wyde PR, Moylett EH, Chetty SN et al (2004) Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by NMS03 in tissue culture assays. Antiviral Res 63 51-59... 

Thus, the interactions between cultured cells and their micro-environment that control the process of cell movement have, as their hub, an adhesive component. Manipulation of cell adhesion in tissue culture assays leads to predictable changes in cell movements. Could the complex process of neural crest cell migration in vivo also have an important adhesive basis First, in favorable in vivo situations where the dynamics of cell movement can be observed, the process of locomotion of crest-derived cells resembles that seen in the simplified cell culture models (see Newgreen,... 

Kogure, K. et al., A tissue culture assay for tetrodotoxin, saxitoxin and related toxins, Toxicon 26, 191, 1988. 

Gallacher, S. and Birkbeck, T.H. A tissue culture assay for the direct detection of sodium channel blocking toxins in bacterial culture supernatants. FEMS Microbiol. Lett., 92, 101, 1992. 

G12. Gross, J., and Lapiere, C. M., Collagenolytic activity in amphibian tissues A tissue culture assay. Proc. Natl. Acad. Sci. USA 48, 1014-1022 (1962). 

A method of looking for specific inhibitors for RNA-dependent RNA polymerase was employed in the detection of a new compound. A rapid plaque assay utilizing hemadsorption for rubella virus has been developed.Tissue culture assays utilizing a continuous concentration gradient gave a direct indication of tissue culture therapeutic ratio. ... 

In order to address the possibility that the light effect might be artificially created by the tissue culture assay, in which the normal retina/RPE apposition is disrupted and the interphotoreceptor retinoid binding protein is washed away, we did experiments in which the only light... 

The intravascular use of fluorochemicals demands the absence of toxicity and carcinogenic, mutagenic, or teratogenic effects. The toxicity of fluorocarbons is largely affected by their purity. Most fluorocarbons are toxic, unless carefully purified. Tissue culture assays are needed to test for toxicity before a fluoro-chemical can be considered for biomedical application. Perfluorodecalin and per-fluorotripropylamine have been used clinically [35,41,87-89]. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

White blood cells, red blood cells and cultured fibroblasts are commonly used to measure enzyme activities, especially for the diagnosis of inherited enzyme abnormalities. Leukocytes may be collected by sedimentation in viscous media such as Fycol. The collection of red cells presents no problem following centrifugation of anticoagulated blood. The assay of enzymes and fibroblasts requires appropriate tissue culture facilities and extensive experience in dealing with cultured human cells. 

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tabacco tissue culture. Physiol Plant 15 473-497... 

A complete assay requires the test material to be investigated at a minimum of three doses together with a positive (untreated) and solvent-only control can be omitted if tissue culture medium is used as a solvent. When two fixation times are used in repeat tests, the positive control is necessary at only one time but the negative or solvent control is necessary at both times. 

Borenfruend, E. and Puemer, J.A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays. J. Tissue Culture Meth. 9 7-9. 

Enzyme assays—both kinetic and end-point radiocoordination of proteins, lipid assays, receptor binding assays and tissue-culture techniques... 

Based on the results from those cell calibration assays, the routine incubation period was set at 3 days. During that interval, some of the cells will proliferate, some will be quiescent but metabolically active, and some will die, which is the case for most primary cell cultures. Because these cells are cultured for periods far shorter than their typical population doubling time (about 6 to 7 days), these cells should not fully adapt to the tissue culture conditions and, therefore, maintain their primary cell phenotype. 

Only after all these exhaustive tests are a few candidates selected for pre-clinical in vivo studies using animal disease models. The current approach is to perform as many tests as possible based on tissue cultures or cell-based assays, as they are less costly and provide results more readily. At the end of this long process is the availability of selected drug candidates with sufficient efficacy and safety required for human chnical trials. 

Tumor Spheroid-Based Migration Assay 1. Appropriate culture medium for the specific tumor cell line(s) under test. 2. 96-well flat-bottomed tissue culture-treated plates. 

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