Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species

Listeria monocytogenes is cytotoxic to the lymphocyte-origin hybridoma Ped-2E9 cell line. The relative cytotoxicity can be calculated by assaying the release of alkaline phosphatase (ALP) from the infected cell line. In this study, a fluorogenic substrate (4-methylumbelliferyl phosphate, MUP) was used to quantify the ALP activity. The assay is 3.5-fold more sensitive than the colorimetric-based assay and requires only 1 h to differentiate virulent from avirulent strains. In addition to various Listeria species, 27 different common foodborne or clinical microorganisms were tested with the fluorescence-based cytotoxicity assay and only six cultures (Bacillus cereus, Citrobacter freundii, Serratia marcescens, Pseudomonas putida, Corynebacterium glutamicum and Micrococcus luteus) showed cytotoxic effects similar to L. monocytogenes. To use this assay as a confirmatory test for virulent L. monocytogenes suspect strains, pure cultures must be isolated from the sample prior to testing.     

Rapid detection of Listeria species by selective impedimetric assay

A selective medium was developed for the detection of Listeria monocytogenes, L. innocua and L. welshimeri by impedimetric assay. Addition of 21 mg l^-1 proflavine hydrochloride and 20 mg l^-1 moxalactam to glucose-enriched nutrient broth suppressed the impedimetric responses of Gram-negative and other Gram-positive test micro-organisms, permitting the quantitative detection of the three Listeria species within 36 h.     

The efficiency and linearity of the radiochromium release assay for cell-mediated cytotoxicity.

The release of radiochromium from EL-4 cells lysed by anti EL-4 peritoneal lymphocytes is a first order decay process with respect to time. The rate of ⁵¹Cr release from damaged cells was independent of the effector: target ratio but was slower from pelleted cells than cells in suspension. We conclude that under normal assay conditions the fraction of chromium released is nearly proportional to the number of cells damaged but find that the proportionality constant is influenced by assay conditions. There is a lag time of 23 ± 5 min before ⁵¹Cr is rapidly released from the cells.     

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.     

A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 microm and 900 microm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario.     

Alkaline phosphatase conductometric biosensor for heavy-metal ions determination

Alkaline phosphatase conductometric biosensors consisting of interdigitated gold electrodes and enzyme membranes have been used for assessment of heavy-metal ions in water. These analytes act as enzyme inhibitors. Enzyme residual activity has been measured in Tris-nitrate buffer without metal preincubation in the presence of Mg²⁺ ions as activator. The results indicate that the toxicity of the various metals tested toward immobilized phosphatase is ranged as follows: Cd²⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Pb²⁺. Detection limits were about 0.5 ppm for Cd²⁺, 2 ppm for both Zn²⁺ and Co²⁺, 5 ppm for Ni²⁺ and 40 ppm for lead ions. In addition, the responses during 10 h were stable (RSD 4%) and a drift of about 7% per day was observed. The storage stability in buffer solution at 4 °C remained stable for more than one month.     

Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.     

Modified enzyme activity assay to determine biofilm biomass

An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm. The assay utilized the nonfluorescent compound L-leucine-beta-naphthylamide (LLbetaN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome beta-naphthylamine. Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer. A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon. The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum. Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay. The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine. While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.     

Sulforhodamine B colorimetric assay for cytotoxicity screening

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.     

An automated potentiometric assay for acid phosphatase

Two flow injection systems for detection of acid phosphatase (ACP) activity have been investigated. In both systems, monofluorophosphate (MFP) as a specific and inexpensive substrate for ACP and LaF₃ membrane-based ion-selective electrode as a potentiometric detector of fluoride ions released in the course of the biocatalytic hydrolysis of MFP were applied. The continuous-flow mode was modified to include a stopped-flow segment, with separate valve control, to increase reaction time and minimize dispersion while increasing system throughput. The bioanalytical utility of these systems was compared for quantifying of ACP activity in human serum standards.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

A gel filtration assay to determine glycogen synthase activity

We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.     

Alkaline phosphatase activity in two planktonic desmid species and the possible role of an extracellular envelope

1. Cosmarium abbreviatum var. planctonicum , a desmid from oligo‐mesotrophic lakes, had a higher maximum alkaline phosphatase activity (APA) and affinity constant under both continuous and pulsed inorganic phosphorus (P_i) limitation than Staurastrum chaetoceras , a desmid predominantly encountered in eutrophic lakes.
2. APA of both species increased when measured in starved cells subjected to pulsed P_i conditions when compared to continuous P_i limitation.
3. The portion of extracellular relative to cellular APA was higher in S. chaetoceras than in C. abbreviatum , indicating that S. chaetoceras secreted the enzymes more readily into its environment.
4. The difference in APA could explain the dominance of C. abbreviatum during competition between these two species under conditions of continuous organic P (P_o) limitation, but not the outcome under a pulsed P_o shortage. The dominance of S. chaetoceras in the latter experiment can, however, be explained by species‐specific P_i‐uptake characteristics.
5. After a saturating pulse of P_i, no increase in P_i in the extracellular mucus layer of C. abbreviatum was found and it was therefore concluded that the mucilage sheath does not store P. However, the sheath could have a main function as an accumulation site of cellular APA, providing the cell with P_i.     

Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin

An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.     

An improved resazurin-based cytotoxicity assay for hepatic cells

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alkaline phosphatase activity as a membrane marker for activated B cells

Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.     

Filtration assay for arachidonic acid release

Arachidonic acid (AA) release is a central message in cell signaling. Fatty acid release is generally assessed by manual sampling of radioactivity release from cells prelabeled with a radiolabeled fatty acid. The assay is laborious, time-consuming, and susceptible to high noise. Here we present a fast and reproducible method for 96-well filter plates and cells in suspension, a method that is best suited for agonist concentration-response studies and, thus, for ligand screening. The method offers tremendous time and effort savings and enables execution of large experimental series previously unattainable for AA release studies.