A first step in visual identification of different cell populations by a modified alkaline comet assay

Using a particular model of apoptosis, we here demonstrate the ability of the comet assay to differentiate between different cell populations. In our study, the natural killer Kurloff cells, used as effector cells, recognize and bind to the tumoral L2C target cells. Formation of such conjugates leads to the death of the target cells by apoptosis, as previously described by different conventional techniques. With the alkaline comet assay, a conjugate could directly be visualized as an association of an undamaged cell joined to a highly damaged cell. The modified comet assay used in this study comprises specific labelling of Kurloff cells with immunomagnetic beads, which are visible as grey-dull spheres against the bright-red staining of nuclear origin on the comet preparation. The use of such labelled effector cells suggest the potential of the comet assay to visually identify different cell populations in an unique test.     

A first step in visual identification of different cell populations by a modified alkaline comet assay

Using a particular model of apoptosis, we here demonstrate the ability of the comet assay to differentiate between different cell populations. In our study, the natural killer Kurloff cells, used as effector cells, recognize and bind to the tumoral L2C target cells. Formation of such conjugates leads to the death of the target cells by apoptosis, as previously described by different conventional techniques. With the alkaline comet assay, a conjugate could directly be visualized as an association of an undamaged cell joined to a highly damaged cell. The modified comet assay used in this study comprises specific labelling of Kurloff cells with immunomagnetic beads, which are visible as grey-dull spheres against the bright-red staining of nuclear origin on the comet preparation. The use of such labelled effector cells suggest the potential of the comet assay to visually identify different cell populations in an unique test.     

A study of MTT-hybrid assay using human bone and soft tissue tumor cells

We investigated a new chemosensitivity test, MTT-hybrid assay, which was a hybrid of MTT colorimetric assay and double-layered soft agar colony assay, using human bone and soft tissue tumor cells. MTT formazan crystals produced by viable cells in the soft agar medium were solubilized by SDS at 60 degrees C. The absorbance (560 nm) is directly proportional to the cell number over a wide range. The absorbance increased in proportion to colonial growth of osteosarcoma cells, while it decreased in a human diploid cell strain in a few days. Drug sensitivity of tumor cells is supposed to be assessed without contaminating normal cells by MTT-hybrid assay in primary tumor samples. Good correlation of IC50 was observed between MTT-hybrid assay and colony assay. The MTT-hybrid assay shows potential value as a rapid predictive test for chemotherapeutic agents in an individual patient.     

The kinetics of interferon production by mouse lymphocytes and its modulating effect of the virus plaqueforming cell assay as a quantitative method to determine activated lymphocytes.

The validity of the virus plaque-forming cell (V-PFC) assay as a means to quantitate stimulated mouse T cells is dependent on the rate and amount of coinciding interferon production which varies with different strains of mice. In addition, the V-PFC assay demonstrates that the kinetics of cellular activation by mitogens initially involves about 0.01% of responding cells and possibly recruitment of other cells which include cells requiring a longer lap period for activation.     

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.     

Capillary cloning of primary human tumor cells: assay miniaturization for drug efficacy testing

The conventional double-layer agar method of cloning human tumor cells requires a substantial number of viable tumor cells and 14-21 days of culture. These prerequisites frequently limit its utility as an assay. In an attempt to circumvent these limitations and to reduce the amount of drug that is needed in the assay, we have further developed and miniaturized the assay in which human tumor cells are cloned in glass microcapillary tubes. Cultures consisted of 50 microliters containing 15,000 nucleated cells in 975 mm capillary tubes which were incubated for seven days. The results from 50 consecutive tumor biopsies resulted in cloning efficiencies, ranging from 0.007% to 1.0% with an overall successful cloning of 88% of all tumors tested and a good linear growth relationship and chemotherapy sensitivity. This miniaturized assay offers distinct advantages for drug efficacy testing including high cloning efficiencies, small tumor sample and drug requirements, quicker assay turnaround time and a general conservancy of reagents and incubator space.     

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

A colorimetric assay for the assessment of cytotoxicity of yeasts

A colorimetric assay for the quantitation of microbial cytotoxicity has been developed using cells from a monocyte-like human cell line (U937), epithelial cells (Hela), and fibroblast-like cells (Vero) as targets. The fraction of surviving cells was determined by their content of the dye neutral red which is retained only by live cells and can be quantitated photometrically after controlled lysis. The neutral red retention assay was at least as sensitive as the 51Cr-release assay; it was considerably less laborious, faster, and avoided handling of radioactivity. Among the different Candida species tested, the highest cytotoxicity was associated with C. albicans and C. tropicalis; a lower degree of cytotoxicity was exhibited by C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. Among the strains of a given fungal species cytotoxicity varied by up to 40%.     

A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication

This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.     

New possibilities of cytostatic drug testing on patient tumor cells by flow cytometry

A new assay for cytostatic drug testing is described which can be automated. Pleural effusions and ascites are cultured as such for one week. Cells of solid tumors are cultured in the patients own serum for the same time. The cells are then stained with the esterase and intracellular pH-indicator dye 1,4-diacetoxy-2,3-dicyano-benzene (ADB) to label vital cells. They are simultaneously stained with propidium iodide (PI) as an indicator for dead cells. Monosized fluorescent latex particles are added as concentration, volume and fluorescence standard. Inflammatory cells can be distinguished in the assay from tumor cells because of their small cell volume. The number of dead and surviving cells is counted by the flow cytometer and a therapeutic index is calculated as ratio between the surviving inflammatory to surviving tumor cells. An important feature of the assay is that the DNA-distribution of the dead cells (e.g. aneuploidy) as well as the functional state of the surviving tumor cells and inflammatory cells can be judged from intracellular esterase activity and intracellular pH.     

Use of the micronucleus assay with exfoliated epithelial cells as a biomarker for monitoring individuals at elevated risk of genetic damage and in chemoprevention trials.

This review summarises the current database on the micronucleus (MN) assay with exfoliated cells (MEC assay) and evaluates the predictive value of this model for the detection of human cancer risks. The MEC test is a cost effective, non-invasive method, in which the formation of MN in exfoliated cells from different organs, such as oral and nasal cavity, bladder, cervix, and oesophagus is used as an endpoint to detect endogenous, lifestyle, occupational and environmental exposures to genotoxins as well as chemoprotection of various compounds in intervention studies. The results suggest that the MN assay might be a useful approach to identify antimutagens which are protective in humans. Based on the comparison of the data from MN experiments with results from epidemiological cancer studies, we conclude that the MEC assay is a useful biomarker for the detection of human cancer risk in organs to which the MEC test can be applied. However, the current data base is not sufficient to draw a firm conclusion on the specificity of this approach.     

In Situ Assay of Hormone-Stimulated Adenylyl Cyclase in 96-Well Microtitration Plates: An Aide to Rapid Identification of Transformed Cell Clones

Abstract

An in situ assay able to detect hormonally stimulated or inhibited adenylyl cyclase (AC) activity on as few as 5,000 cells/well has been developed. In addition the assay monitors phosphatase activity which serves as a marker for cell density. Cells are plated in replicate wells at least one day before the assay, and the medium containing AC reagents, an ATP regenerating system, a PDE inhibitor, additives that regulate receptors and/or G proteins, and 5 mM pnitrophenyl phosphate (pNPP), plus Tris buffer to pH 7.5 is added. The hypotonic medium causes permeabilization of cells without massive lysis. After stopping the reaction with 100 μl of a solution with ATP, cAMP and SDS, the color indicating phosphatase activity is quantified by an ELISA reader, and AC activity measured by standard methods. At proper cell density (pNPP hydrolysis) the assay shows proportionality up to 2 hours. The assay is particularly useful in transfection experiments. As few as 50,000 cells can be plated and identified as receptor “positive” or receptor “negative”. The assay was key to our cloning of the V2 AVP receptor. The assay accelerated the preparation of stable cell lines with LH, FSH, adrenergic and serotonin IDβ/1B and IE receptors. It should also be useful in studies in which the transfected cDNA encodes the adenylyl cyclase proper.     

Green fluorescent protein: A novel viability assay for cryobiological applications

Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ. Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly. Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues. As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability. To the authors' knowledge, this is the first use of GFP in cryobiology applications. Intracellular GFP fluorescence was evaluated following slow freezing. Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath. Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr). A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97. A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay. Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions. The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate.     

Cholera Toxin Inhibits Lethal Hit Stage of Natural Killer Cell-Mediated Cytotoxicity

Cytolytic process which was affected by cholera toxin (CT) resulting in the loss of natural killer (NK) cell activity was analyzed. Conjugate formation assay, membrane phospholipid methylation assay and serine esterase (granzyme A) release assay were used to determine the stage of the CT-induced inhibition of NK cell-mediated cytotoxicity. A human NK cell line YT cell-mediated cytotoxicity was completely abolished by CT pretreatment or addition of CT to the assay system. The conjugate formation assay revealed that the binding between YT cells and target cells was not affected by CT. The defined triggering stage which is coupled with membrane phospholipid methylation was not affected by CT treatment, either. On the other hand, the lethal hit stage which is represented by serine esterase (SE) release was completely inhibited by CT treatment of YT cells. Therefore, CT inhibits the stage after binding and triggering—i.e., lethal hit stage of NK cell-mediated cytotoxicity. The results also suggest that there exists a CT-sensitive negative cytotoxic signal transduction pathway as well as usual positive signal transduction pathway and these pathways might cross talk each other in the NK cell cytotoxic process.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Characterization of the in vitro interaction of PNAhi lymphocytes with the bone marrow and hepatic asialoglycoprotein receptors

We have previously identified an in vivo interaction between circulating PNAhi lymphoid cells and the hepatic asialoglycoprotein receptor, which results in a protracted liver sequestration of these cells. An in vitro frozen section binding assay was developed to study the interaction of PNAhi cells with the receptor in more detail. This assay confirmed that the sequestration of PNAhi lymphoid cells by the liver was mediated by the asialoglycoprotein receptor, as binding was inhibitable by coincubation with galactose, asialoglycoproteins, chelation of divalent cations, or a specific anti-asialoglycoprotein receptor antiserum. This frozen section binding assay was utilized to demonstrate the existence of a bone marrow asialoglycoprotein receptor which was found to be capable of binding to PNAhi lymphocytes or asialoglycoproteins bound to synthetic substrates. We further established that the bone marrow receptor differed both functionally and antigenically from its hepatic analogue.     

Dominant lethal cell mutants detected by the autoradiographic assay for exotoxin A resistance

The autoradiographic assay (AR assay) for P. aeruginosa exotoxin A (PE) resistance in cultured mouse fibroblasts detects mutants able to synthesize proteins in the presence of the toxin, presumably due to mutations in the structural gene for elongation factor 2 (EF-2). Detection by the AR assay of PER cells is independent of their ability to divide. The frequencies of both spontaneous and mutagen-induced PER cells are higher than those detected by the conventional colony assay. Examination of phenotypic expression times in the PER cells, and of their in situ proliferation, reveals that this higher sensitivity of the AR assay is due to its ability to detect cells in which the PER mutation prevents proliferation, thus escaping detection by the colony assay. Expression of the mutant phenotype in the PER cells detected in the AR assay after mutagenesis with ethyl methanesulfonate (EMS) follows a pattern similar to that observed in the colony assay, reaching a maximum in 3 days, and then remaining constant for at least 4 more. After treatment with X rays (which fail to induce PER mutants in the colony assay), the frequency of PER cells detected in the AR assay also reaches a maximum on day 3, but then declines sharply, returning to the spontaneous level on day 7. In the absence of PE, the majority of the spontaneous or mutagen-induced PER cells detected in the AR assay are either incapable of dividing at all, or capable of undergoing a limited number of cell divisions to produce micro-colonies. Only few of them may continue to grow into 'full-size' colonies comparable to those detected in the colony assay. In the presence of the toxin, the proportion of PER cells which are able to divide is even smaller, and that of cells able to form full-sized PER colonies detectable in the AR assay is comparable to the results obtained in the conventional colony assay. We presume that the lethality of the PER mutations in the cells detected by the AR assay is due to abnormal protein synthesis resulting from the same mutational change that made these cells resistant to PE. While incapable of supporting colony formation, and hence detection by the colony assay, such abnormal protein synthesis still allows the detection of the mutant cells by the AR assay.