A new electro-optical approach to rapid assay of cell viability

A new electro-optical (EO) approach was developed and applied to rapidly assay cell viability by using phage M13K07. Since phage M13K07 can replicate only in living bacteria and cannot replicate in the presence of inhibitors, the difference between the EO signals obtained in the presence and absence of the phage can be used as an important factor for evaluating cell viability. Variation in the electrophysical parameters of Escherichia coli XL-1 during its interaction with phage M13K07 was studied under exposure of the cells to various inhibitors of cellular metabolism. Significant changes in the EO signal were found during incubation of living E. coli cells with phage M13K07. At the same time, no changes were recorded during cell incubation with the phage after pretreatment of E. coli XL-1 cells with sodium azide, carbonyl cyanide 3-chlorophenyl hydrazone, chloramphenicol, and kanamycin. This finding can be explained by the decrease in the number of living cells in the culture after preliminary incubation with the chemical agents, and it was confirmed by colony counts by conventional plating onto solid LB medium before and after treatment of the cells with the inhibitors. The EO approach can be used as a rapid method for evaluation of the inhibitory effects of various chemical agents and drugs, and it has the potential for the study of the molecular mechanisms underlying cell death.     

Dystrophin: a sensitive and reliable immunochemical assay in tissue and cell culture homogenates.

A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3-12 micrograms) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques.     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.     

A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts

Summary A simple, reliable and sensitive assay for alpha-amylase activity is reported, together with its theoretical derivation, that overcomes many of the problems encountered with other assays, especially when attempting to assay alpha-amylase activity in crude cell extracts or culture supernatants. The method relies on the reduction in turbidity that occurs upon digestion of a starch suspension with alpha-amylase. The initial rate of decrease in turbidity is shown to be proportional to a wide range of enzyme concentrations, permitting a rapid spectrophotometric and kinetic determination of alpha-amylase activity.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

An oxygen electrode assay to determine autoclaved-induced toxicity in tissue culture media

An oxygen electrode was used to assay cyanide-induced oxygen consumption of 8 sugars following autoclaving and the results correlated with the effects of the same sugars (reported by de Lange, S. Afr. J. Bol. 55, 1, 1989) on pollen germination and pollen tube growth. Of the sugars tested, fructose was the least stable, producing a degradation product that resulted in a 40-fold higher consumption of oxygen than sucrose. The use of cyanide-induced oxygen uptake makes it possible to control more carefully the conditions of preparation of sterile media by autoclaving.     

A new method to determine initial viability of entrapped cells using fluorescent nucleic acid staining

Entrapped bacterial cells are widely used in several biotechnological applications. Cell entrapment procedures are known to affect the viability of bacterial cells. To determine the effect of entrapment procedures on viability of bacterial cells, dissolution of the entrapment matrices using chelating agents or heat is required immediately after the entrapment is completed. Chelating agents and heat applied in the matrix dissolution reduce cell viability and in turn hinder accurate quantification of viable cells. In this study, a method to determine the effect of entrapment procedure on bacterial cell viability which involves entrapping cells directly onto glass slides was developed. The developed method showed less viability reduction than the methods requiring matrix dissolution. The percentage of live cells in the culture before entrapment ranged from 54% to 74%, while the percent of live cells after entrapment determined by the developed method was 39-62%.     

A new chronological survival assay in mammalian cell culture

c-Abl is activated by oxidative stress but its precise function in cell response to this stress is elusive. Studies of c-Abl-/- osteoblasts revealed that c-Abl played a negative role in the induction of peroxiredoxin I (Prx I, Prdx I), an anti-oxidant protein with tumor suppression activity, but it provided a protective role against oxidative stress. In contrast, Atm, a signaling molecule that interacts with c-Abl and is required for c-Abl activation, served a totally different function. The significance of these findings is discussed here in the context of aging and tumorigenesis and their links to reactive oxygen species.     

Arachidonic acid release in cell lines transfected with muscarinic receptors: a simple functional assay to determine response of agonists.

Muscarinic agonists stimulated arachidonic acid release from 10- to 32-fold in Chinese hamster ovary (CHO) cells transfected with muscarinic M1, M3 and M5 receptor subtypes. Muscarinic agonists liberated arachidonic acid from the cAMP-coupled M2 and M4 cells only in the presence of ATP. Partial agonists were less efficacious at liberating arachidonic acid than full agonists. The ability of muscarinic agonists to liberate arachidonic acid and stimulate phosphoinositide hydrolysis in the same CHO M1, M3 and M5 cells was well correlated; however, partial agonists were more efficacious at stimulating phosphoinositide hydrolysis than arachidonic acid release. The efficacy and potency of 13 muscarinic agonists to liberate arachidonic acid was characterised. Influx of external calcium was required for arachidonic acid release even after initiation of agonist-induced release. It is concluded that arachidonic acid release is a simple assay suitable for evaluation of muscarinic agonists, antagonists and the flux of external calcium into cells.     

A tool for modeling strategic decisions in cell culture manufacturing

The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed. A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility. The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study. In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs. The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option.     

Loss of viability correlates with altered acid phosphatase activity in suspended strawberry cell fractions after excessive agitation

Acid phosphatase (EC 3.1.3.2.) activity in all fractions and growth phases of strawberry cell culture was 3-fold higher in the excessively agitated cells compared to a control. The stressed cells lost 9% of viability in the lag phase. Concomitantly the enzyme activity ratio of the lysosome fraction to the crude organelle mixture decreased (-33%) whereas the activity ratio of the cytosol fraction to the crude organelle mixture increased (+30%). This change in the enzyme activity of the ratio of the fractions was not observed in the exponential and stationary phase where the loss of viability was only 4 to 5%.     

Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ

A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.     

Improvement of a Vero cell assay to determine diphtheria antitoxin content in sera

Diphtheria antitoxin content in sera were determined automatically in Vero cell assay by spectrophotometric determination of the equivalence point between toxin and antitoxin followed by computer analysis of absorption values. The method was more accurate than visual reading and made handling of many samples easy.     

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Objectives

Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes.

Results

A correlation between evaporation and the concentration of Na⁺ was found (R²?=?0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na⁺ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7?±?6.7% without volume corrections to 6.9?±?6.5% with volume corrections.

Conclusion

A procedure was established to indirectly measure evaporation through a correlation with the level of Na⁺ ions in solution and deriving a simple formula to account for liquid losses.

     

A quantitative cytochemical assay for osteoclast acid phosphatase activity in foetal rat calvaria

Summary Acid phosphatase activity is prominent in osteoclasts (bone resorbing cells) and has been implicated in the process of bone resorption, although its precise role is not understood. To study the distribution and activity of this enzyme, a quantitative cytochemical method has been developed using undecalcified fresh frozen sections of foetal rat calvariae. Sections were allowed to react with 3mm naphthol ASBI phosphate at pH 5.0, and the reaction was stopped by rinsing in ice-cold tap water containing 50mm sodium fluoride. The reaction product was post-coupled to Fast Garnet at 4°C. The absorbance of areas of reaction product in the cytoplasm was measured using scanning and integrating microdensitometry. The initial velocity rate was maintained for up to 2 min at pH 5.0 with a substrate concentration of 3mm and a section thickness of 5 µm. Under these conditions reaction product was localized to osteoclasts and the surface of bone matrix beneath these cells. Activities in osteoblasts and chondrocytes were negligible. Osteoclastic acid phosphatase was almost totally inhibited by 10mm fluoride and reduced by 70% by 100mm tartrate.     

A high-molecular weight complex with acid phosphatase activity in human breast cancer

Summary The presence of a high-molecular weight complex with acid phosphatase activity in the cytosol of human mammary tumors is reported. This complex appeared in the cytosol after tissue homogenization in the presence of dithiotreitol, with or without Triton X-100 and at acidic or neutral pH. Upon gel electrophoresis, this fraction showed only one band of enzyme activity which did not enter the fine pore gel. Lubrol or n-butanol had no apparent effect on this complex, and 8 M urea or 2% sodium dodecyl sulfate did not disaggregate this large molecule. After purification by gel filtration, ammonium sulfate precipitation and ion-exchange chromatography an apparent molecular weight or 10⁶ was measured. It hydrolyzed typical acid phosphatase substrates such as p-NPP and -NP, but also ATP and PPi. Only 44% inhibition was observed with L-(+)tartrate and it was still 40% active after 1 hr incubation at 60 °C. Reduction in the presence of SDS yielded several polypeptide bands. It was also detected in some samples of normal mammary tissues, but not in normal human placenta or liver.Abbreviations AP acid phosphatase - C-AP high-molecular weight complex with acid phosphatase activity - SDS sodium dodecyl sulfate - p-NPP p-nitrophenyl phosphate - -NP -naphthyl phosphate - enzyme code number acid phosphatase or ortophosphoric monoester phosphohydrolase (EC 3.1.3.2)     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD