Lactate dehydrogenase cell culture

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

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). 

Another approach to overcome assay chemistry incompatibility is splitting a sample into two different containers. If the cell population releases a marker into the surrounding environment, a small aliquot of culture medium can be sampled and moved to a different assay plate to segregate assay chemistries. Figure 6.11 shows the measurement of lactate dehydrogenase released from a... 

Koh JY, Choi DW (1987) Quantitative determination of glutamate mediated cortical neurontil injury in cell culture by lactate dehydrogenase efflux assay. J Neurosd Methods 20 83—90 Koh JY, Choi DW (1994) Zinc toxicity on cultured cortical neurons involvement of N-methyl-D-aspartate receptors. Neuroscience 60 1049-1057... 

The most common assay systems consist of cell cultures that are treated with the drug in the culture medium. Toxicity is commonly assessed by release of intracellular enzyme, for example, lactate dehydrogenase or aspartate transaminase, into the culture medium or by other indicators such as decreases in the rate of radiolabeled amino acid or nucteotide precursor incorporation into macromolecules. A decrease in the intracellular uptake of the vital dye, neutral red, has also... 

Legrand C, Bour JM, Jacob C, Capiaumont J, Martial A, Marc A, Wudtke M, Kretzmer G, Demangel C, Duval D Hache J (1992) Lactate dehydrogenase (LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells as marker. Journal of Biotechnology 25 231-243. 

Cell lysis generally becomes important when viability is low. Examples include continuous culture (with or without cell retention) at low dilution rates and the late stationary and death phases of batch culture. Cell lysis is also important in stirred reactors with very high agitation rates. In order to quantify cell growth parameters under these conditions, the lysed cells must be accounted for. One way to do this is to measure the amount of the cytosolic enzyme lactate dehydrogenase (LDH) released into the medium (see Chapter 2, section 2.5). A procedure for measuring LDH activity is described in Chapter 4, section 4.7. 

The extent of cell death is determined by measuring the cells unable to exclude the Trypan blue dye. In prolonged continuous culture or in unfavourable environments the additional phenomenon of cell lysis may also occur. The number of cells that have lysed can be determined by measuring the lactate dehydrogenase (LDH) concentration released into the medium (see Chapter 2, section 2.5). Adding the number of visible and lysed dead cells yields an evaluation of the total rate of cell death. 

Goergen JL, Marc A Engasser JM (1993) Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology 11 189-195. 

Studies of ibuprofen and other NSAIDs have produced toxic effects at concentrations 10 times therapeutic in cultured hepatocytes. No adverse effect on cell survival was noted at therapeutic concentrations of ibuprofen in this model, although increases in lactate dehydrogenase leakage were prominent. 

N-Acetyl-B-D-glucosaminidase (N-Ac-Glu) 6 3), B-Gl ucuroni-dase (B-Glu) 4)and lactate dehydrogenase (LDH)(25lThe latter enzyme is located in the cytoplasm and is not actively secreted (as the other two enzymes are) but only set free if the cell membrane is destroyed the LDH liberated informs about viability of cell cultures or cytotoxic effects on macrophages of the substance tested. 

The nephrotoxicity of 16 continues to generate considerable interest. Orellanine was highly toxic to mice (LD50 = 12.5 mg/kg i.p.)[101] and caused interstitial nephritis and tubular necrosis in mouse kidney [102], A summary of 16-induced changes in renal function and morphology has been reported [103]. In LLC-PKi renal epithelial cell cultures, 16 decreased the activity of alkaline phosphatase and lactate dehydrogenase, and decreased the incorporation of H-leucine and H-thymidine [104]. Orellanine was a noncompetitive inhibitor of renal alkaline phosphatase, but a competitive inhibitor of the intestinal and placental enzymes [105]. In canine kidney MDCK cell cultures, 16, or a metabolite of 16, inhibited protein, RNA and DNA synthesis [106]. 

Fig. 1. Time course of cell death parameters in tumor necrosis factor (TNF)-induced apoptosis. Human hepatoma cells (HepG2) were incubated in the presence of 400 nM actinomycin D plus 1.6 ng/ml recombinant human TNF (rhTNF)-a. MTT-dye reduction (parameter of mitochondrial function), lactate dehydrogenase (LDH) release (parameter for cell membrane rupture), and DNA fragmentation (parameter associated with typically apoptotic nuclear changes) were determined over a time period of 24 h. As is characteristic of apoptotic cell death, DNA fragmentation always preceded the other death parameters. Similar findings have been obtained in primary hepatocyte cultures and murine livers...

Under aerobic conditions the long slender forms metabolize glucose quantitatively to pyruvate. Trace amounts of CO2 and sometimes glycerol have been reported as end products. However, CO2 may result from a spontaneous decarboxylation of pyruvate, whereas the glycerol most likely results from a partial anaerobiosis of the cells during culture or incubation. Pyruvate cannot be converted into lactate because of the absence of lactate dehydrogenase enzymatic decarboxylation of pyruvate does not occur in these forms due to the absence of pyruvate decarboxylase. 

The strain used in this study was Thermoanaerobacter BGILI, which is a lactate dehydrogenase-defieient mutant of the thermophilic anaerobic bacterial strain BGl. The strains Thermoanaerobacter BGl and BGILI have been deposited in the German Collection of Microorganisms and Cell Cultures as patented strains. 

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