Alamar Blue Dye Research Articles

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

Identification and Functional Characterization of Three Chicken Cathelicidins with Potent Antimicrobial Activity

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 microm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 microm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.

Neutralizing Antibodies and Persistence of Immunity following Anthrax Vaccination

Anthrax toxin consists of protective antigen (PA) and two toxic components, lethal factor (LF) and edema factor (EF). PA binds to mammalian cellular receptors and delivers the toxic components to the cytoplasm. PA is the primary antigenic component of the current anthrax vaccine. Immunity is due to the generation of antibodies that prevent the PA-mediated internalization of LF and EF. In this study, we characterized sera obtained from vaccinated military personnel. Anthrax vaccine is administered in a series of six injections at 0, 2, and 4 weeks and 6, 12, and 18 months, followed by annual boosters. The vaccination histories of the subjects were highly varied; many subjects had not completed the entire series, and several had not received annual boosters. We developed a simple colorimetric assay using alamarBlue dye to assess the antibody-mediated neutralization of LF-mediated toxicity to the J774A.1 murine macrophage cell line. Recently vaccinated individuals had high antibody levels and neutralizing activity. One individual who had not been boosted for 5 years had low immunoglobulin G antibody levels but a detectable neutralization activity, suggesting that this individual produced low levels of very active antibodies.

Effect of gemcitabine on the expression of apoptosis-related genes in human pancreatic cancer cells

To investigate the expression of genes involved in the gemcitabine-induced cytotoxicity in human pancreatic cancer cells. A human pancreatic cancer cell line, PANC-1, was cultured. 1x10(4) PANC-1 cells were plated in 96-well microtiter plates. After being incubated for 24 h, gemcitabine was added to the medium at concentrations ranging 2.5-1000 mg/L. The AlamarBlue dye method was used for cell growth analysis. DNA fragmentation was quantitatively assayed using a DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit. PAP and TP53INP1 mRNA expression was determined using the reverse transcription-polymerase chain reaction with semi-quantitative analysis. The expression of GSK-3beta and phospho-GSK-3beta proteins was examined with Western blot analysis. The IC50 for the drug after a 48-h exposure to gemcitabine was 16 mg/L. The growth of PANC-1 cells was inhibited by gemcitabine in a concentration-dependent manner (P<0.0001) and the cell growth was also inhibited throughout the time course (P<0.0001). The DNA fragmentation rate in the gemcitabine-treated group at 48 h was 44.7%, whereas it was 25.3% in the untreated group. The PAP mRNA expression was decreased after being treated with gemcitabine, whereas the TP53INP1 mRNA was increased by the gemcitabine treatment. Western blot analysis showed that phospho- GSK-3beta (ser9) was induced by the gemcitabine treatment. Gemcitabine suppresses PANC-1 cell proliferation and induces apoptosis. Apoptosis is considered to be associated with the inhibition of PAP and GSK-3beta, and the activation of TP53INP1 and pospho-GSK-3beta (ser9).

Development of a Cell-Based High-Throughput Specificity Screen Using a Hepatitis C Virus-Bovine Viral Diarrhea Virus Dual Replicon Assay

The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.

Perfluorocarbon emulsion improves cerebral oxygenation and mitochondrial function after fluid percussion brain injury in rats.

Cerebral ischemia is a common secondary sequela of traumatic brain injury (TBI). Experimental models of stroke have demonstrated reductions in ischemia after perfluorocarbon (PFC) administration; however, there are no published reports of PFC efficacy after TBI. The current study analyzed the effect of the PFC emulsion Oxygent (AF0144; Alliance Pharmaceutical Corp., San Diego, CA) on cerebral oxygenation, mitochondrial redox potential, and free radical formation after lateral fluid percussion injury. After fluid percussion injury, five 2.25 ml/kg doses of PFC or saline were administered to rats breathing 100% O(2), and oxygen tension was recorded. In a second experiment, a single bolus (11.25 ml/kg) of PFC or saline was given after injury, and redox potential and free radical formation were measured at 1 or 4 hours with Alamar blue dye and dihydrorhodamine 123, respectively. Cerebral oxygen tension was significantly increased in both injured and sham animals treated with 11.25 ml/kg of PFC as compared with saline (P < 0.05). Likewise, PFC significantly increased mitochondrial redox potential as compared with saline at 4 hours after injury (P < 0.01). Mitochondrial peroxynitrite and peroxide production also increased with the administration of PFC (P < 0.05). The current study demonstrates that a PFC emulsion can significantly increase cerebral oxygenation after TBI and enhance mitochondrial function at 4 hours after injury as compared with saline. This study demonstrates a new therapeutic potential for PFC to enhance cerebral oxygenation and aerobic metabolism after TBI. However, the increased free radical formation with high-dose PFCs suggests the need for further studies combining PFCs with free radical scavengers.

Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases.

The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs. The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB. A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL. The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye. None of the aqueous extracts had antimycobacterial activity. Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis. Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract. The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity. Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates. In contrast, it did not inhibit the growth of non-tuberculous mycobacteria. The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL. This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB.

Colorimetric In Vitro Evaluation of T-Cell Cytotoxicity

We propose a new colorimetric method for evaluation of specific T lymphocyte cytotoxicity based on the capacity of viable cells to incorporate alamar blue dye, an indicator of cell metabolic activity. This method is effective for highly specific strains of cytotoxic T lymphocytes and for primary T killers formed in vivo and constituting a small part of analyzed cell population. This test is a simple nonradioactive method for evaluating specific cellular cytotoxicity exhibiting sufficient sensitivity for evaluation of this parameter in the presence of effector cells.

Delivery of Transforming Growth Factor-β2-Perturbing Antibody in a Collagen Vehicle Inhibits Cranial Suture Fusion in Calvarial Organ Culture

Abstract Objective: To determine whether antibody perturbation of Tgf-β, delivered in a collagen gel, could inhibit cranial suture fusion. Design: Attachment and proliferation of osteoblasts cultured on a collagen gel with or without anti-Tgf-β2 antibody were determined by AlamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, collagen gel with and without anti-Tgf-β2 antibody was injected subperiosteally over the posterior frontal suture of postnatal day 15 rat calvariae. A quantitative analysis of suture fusion was used to measure suture bridging in histological serial sections at various time points. Results: Attachment and proliferation for cells cultured on collagen gel with anti-Tgf-β2 antibody were similar to collagen gel controls. Although proliferation was lower than on tissue culture plastic, cells treated with anti-Tgf-β2 antibody maintained an osteoblastic morphology. After 7, 10, and 15 days in organ culture, anti-Tgf-β2 antibody treatment caused ...

Delivery of transforming growth factor-beta2-perturbing antibody in a collagen vehicle inhibits cranial suture fusion in calvarial organ culture.

To determine whether antibody perturbation of Tgf-beta, delivered in a collagen gel, could inhibit cranial suture fusion. Attachment and proliferation of osteoblasts cultured on a collagen gel with or without anti-Tgf-beta2 antibody were determined by AlamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, collagen gel with and without anti-Tgf-beta2 antibody was injected subperiosteally over the posterior frontal suture of postnatal day 15 rat calvariae. A quantitative analysis of suture fusion was used to measure suture bridging in histological serial sections at various time points. Attachment and proliferation for cells cultured on collagen gel with anti-Tgf-beta2 antibody were similar to collagen gel controls. Although proliferation was lower than on tissue culture plastic, cells treated with anti-Tgf-beta2 antibody maintained an osteoblastic morphology. After 7, 10, and 15 days in organ culture, anti-Tgf-beta2 antibody treatment caused a reduction in the percent bridging of posterior frontal sutures, compared with controls. Sutures exposed to anti-Tgf-beta2 antibody and fibroblast growth factor-2 concurrently did not show an inhibition of bony bridging. These results support previous reports suggesting a role for Tgf-beta2 in cranial suture fusion. In cell culture the collagen gel, both with and without anti-Tgf-beta2 antibody, promoted similar osteoblast attachment, proliferation, and osteoblastic morphology. In organ culture anti-Tgf-beta2 antibody was delivered in a bioactive state via a collagen gel to inhibit cranial suture fusion. Also, the results suggest that the inductive effect of fibroblast growth factor-2 is not dependent on Tgf-beta2 activity. Together, these results provide further support for the role of Tgf-beta2 in cranial suture fusion.

Fibroblast growth factor 2 induces increased calvarial osteoblast proliferation and cranial suture fusion.

Craniosynostosis has been associated with fibroblast growth factors (FGFs) and their receptors. The purpose of this study was to quantitatively determine the effect of FGF2 on rat calvarial osteoblasts and a rat cranial suture formation model. Fetal rat calvarial osteoblasts were cultured with and without FGF2. Cell attachment and proliferation was determined by alamar Blue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, postnatal day 15 rat calvariae with dura mater were placed in serum-free media with and without FGF2. A unique quantitative analysis of suture fusion was developed by obtaining measurements of suture bridging in histological serial sections at progressive stages of fusion. Attachment for cells treated with FGF2 was similar to control. In contrast, proliferation was higher for cells treated with FGF2 while maintaining an osteoblastic morphology. After 5 days in organ culture, FGF2-treated posterior frontal sutures showed a dramatic increase in fusion, compared with untreated controls. This increased fusion was maintained throughout days 7 and 10 in culture. Also, fusion was enhanced on the dural side of the suture, as is normally observed in vivo, and the normal tissue architecture was maintained. These results indicate that FGF2 can promote rat osteoblast attachment and normal cell morphology as well as induce cell proliferation. In calvarial organ culture, FGF2 treatment produced an enhanced suture fusion. These results provide further support for a critical role for FGF2 in cranial suture development. These studies also present a new quantitative approach to evaluating the effect of suture-perturbing growth factors on cranial suture fusion.

The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro.

Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-beta-D-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as Candida albicans, its effect on Aspergillus fumigatus is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with C. albicans. For A. fumigatus, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of A. fumigatus with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within A. fumigatus hyphae are killed when they are exposed to caspofungin.

Fibroblast Growth Factor 2 Induces Increased Calvarial Osteoblast Proliferation and Cranial Suture Fusion

Abstract Objective: Craniosynostosis has been associated with fibroblast growth factors (FGFs) and their receptors. The purpose of this study was to quantitatively determine the effect of FGF2 on rat calvarial osteoblasts and a rat cranial suture formation model. Design: Fetal rat calvarial osteoblasts were cultured with and without FGF2. Cell attachment and proliferation was determined by alamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, postnatal day 15 rat calvariae with dura mater were placed in serum-free media with and without FGF2. A unique quantitative analysis of suture fusion was developed by obtaining measurements of suture bridging in histological serial sections at progressive stages of fusion. Results: Attachment for cells treated with FGF2 was similar to control. In contrast, proliferation was higher for cells treated with FGF2 while maintaining an osteoblastic morphology. After 5 days in organ culture, FGF2-treated posterior frontal suture...

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

Detection of complement-mediated antibody-dependent bactericidal activity in a fluorescence-based serum bactericidal assay for group B Neisseria meningitidis.

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group B Neisseria meningitidis in both human and mouse antisera.

In vitro antimycobacterial activities of Physalis angulata L

The HIV-tuberculosis co-infection has caused an impact on tuberculosis epidemiology all over the world and the efficacies of the therapeutic schemes traditionally prescribed in the treatment of tuberculosis, such as isoniazid, rifampicin and pyrazinamide, have decreased due to the appearance of multidrug-resistant M. tuberculosis strains (MDR). This work is part of research on natural antimicrobial agents from plant extracts through bioassay-guided fractionation, by in vitro determination of the minimum inhibitory concentration (MIC) using the microdilution method with Alamar blue oxidation-reduction dye. Crude CHCl3 Physalis angulata extracts and physalin-containing fractions displayed antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium malmoense and Mycobacterium intracellulare.

Fluorometric assessment of In vitro antidermatophytic activities of antimycotics based on their keratin-penetrating power.

Keratin particles impregnated with amorolfine or clotrimazole in serial doubling dilutions (64 to 0.125 microg/ml) were used to evaluate the activities of these agents against 20 isolates each of Trichophyton mentagrophytes and Trichophyton rubrum in a yeast carbon broth medium incorporating Alamar Blue dye. The proposed MIC with keratin impregnation (MIC(K)) is defined as the lowest concentration of an agent used to impregnate keratin particles that effects a fluorescence-based fungal growth quotient of 0.05 or less. The conventional colorimetric and visual MICs of amorolfine for the dermatophytes, </=0.03 microg/ml for T. mentagrophytes and </=0.063 microg/ml for T. rubrum, were approximately half of those of clotrimazole for the same isolates. The superiority of the MIC(K)s of amorolfine for isolates of T. mentagrophytes (2.0 microg/ml; range, 0.5 to 8.0 microg/ml) and T. rubrum (4.0 microg/ml; range, 2. 0 to 8.0 microg/ml) over those of clotrimazole (32 microg/ml [range, 8.0 to >64 microg/ml] and 64 microg/ml [range, 16 to >64 microg], respectively) may indicate the strong in vivo antidermatophytic activity of amorolfine as a topical agent. The new antidermatophytic susceptibility testing procedure has potential clinical utility for the in vitro screening of agents for use in the topical treatment of superficial mycoses.

A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue®

A quantitative colorimetric assay using the oxidation–reduction indicator Alamar Blue® was developed to measure cytotoxicity of compounds against the protozoan parasite Leishmania. Absorbance increased linearly with the plating density of promastigotes of L. major MRHO/IR/76 vaccine strain up to at least 2.5×10 6 cells/ml when parasites were incubated for 72 h in the presence of 10% Alamar Blue®. The 50% effective dose values of common drugs (amphotericin B, pentostam and paromomycin) obtained by this assay were in the same range as previously determined by other methods. The Alamar Blue® assay permits a simple, reproducible and reliable method for screening antileishmanial drugs.

Assessment of Initial Damage and Recovery Following Exposure of MDCK Cells to an Irritant

The ability of Madin–Darby canine kidney cells (MDCK ) to form cell tight-junctions and orientate correctly on porous membranes has been exploited to model corneal barrier function. While the ability to monitor recovery profiles has not yet been included in a prevalidation trial, its inclusion facilitates the prediction of potential adverse reactions. Combining a viability assay (Alamar blue reduction) with the fluorescein leakage assay, the chemical effects on cell membrane and adhesion molecules can be distinguished. The dose–response curves obtained with Tween 20, isopropanol and benzalkonium chloride indicate that Tween 20 at 200 mg/ml caused a 50% decline in Alamar blue reduction and fluorescein retention, while 100 mg isopropanol/ml and 0.1 mg benzalkonium chloride/ml cause similar effects. The more severe the initial damage the longer the recovery period. If a 20% increase in fluorescein leakage gave recovery in 48 hours, 50% gave marginal recovery in 96 hours. Comparable effects were noted for the restoration of the ability to reduce the Alamar blue dye. Resolution of the damaging effects of benzalkonium chloride and isopropanol took longer than for Tween 20, as is the case in the rabbit in vivo.

The Use of Simultaneous Fluorescence and Differential Phase Confocal Microscopy to Study Alamar Blue™ Reduction in an Epithelial Cell Line

The Alamar Blue™ reduction assay is used as an indicator of cellular viability in in vitro tests for the prediction of ocular irritancy. Alamar Blue itself has a low cytotoxicity, so repeat exposure and recovery studies can be performed on the same cells. This paper contains the results of a preliminary investigation of interactions between the Alamar Blue dye and a confluent monolayer of epithelial Madin-Darby canine kidney cells. This was performed by using an experimental fluorescence microscope and differential phase confocal microscope designed for studying live samples in vitro. The initiation of Alamar Blue reduction to its fluorescent product did not occur at the same time in all cells, but started in a small number and spread progressively through their immediate neighbours. There was clear localisation of the reduced (fluorescent) Alamar Blue within the nuclei and other organelles.