The Extensive and Expensive Impacts of HEp-2 [HeLa], Intestine 407 [HeLa], and Other False Cell Lines in Journal Publications.

Cell lines are essential models for biomedical research. However, they have a common and important problem that needs to be addressed. Cell lines can be misidentified, meaning that they no longer correspond to the donor from whom the cells were first obtained. This problem may arise due to cross-contamination: the accidental introduction of cells from another culture. The contaminant, which is often a rapidly dividing cell line, will overgrow and replace the original culture. The end result is a false cell line, also known as a misidentified or imposter cell line. False cell lines may come from an entirely different species, tissue, or cell type than the original donor. If undetected, false cell lines produce unreliable and irreproducible results that pollute the biomedical literature and threaten the development of reliable drug discovery and meaningful patient treatments.The goal of this study was to ascertain how widespread this problem is and how it affects the literature, as well as to estimate how much funding has been used to produce pools of scientific literature of questionable value. We focus on HEp-2 [HeLa] and Intestine 407 [HeLa], two false cell lines that are widely used in the scientific literature but were shown to be cross-contaminated in 1967. These two cell lines have been used in 8497 and 1397 published articles and extensively described as laryngeal cancer and normal intestine, respectively, rather than their true identity: the cervical cancer cell line HeLa. Discussed are tools, approaches, and resources that can address this issue-both retrospectively and prospectively.

MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells' Migration in a Parallel-Plate Flow Chamber.

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells' adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells' adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells' adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.

A reporter cell line for real-time imaging of autophagy and apoptosis.

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.

Adhesive cell patterning technique using ultrasound vibrations.

This paper investigated an ultrasound vibration cell patterning technique. The ultrasound cell culture dish consisted of a culture dish with a glass bottom and a glass disc with a piezoelectric ring that generated a resonance flexural vibration mode on the bottom of the dish. The growth of HeLa cells on the dish was observed under ultrasound excitation for 24 h. Large ultrasound vibrations on the dish inhibited the cell growth. The acoustic field was predicted with finite element analysis and it was found that the cell growth depended strongly on both the acoustic field in the culture medium and the vibration distribution of the dish. The ultrasound vibrations did not affect the viability of the cells, and the cell growth could be controlled by the flexural vibration of the cultured dish.

Genetic Load and Potential Mutational Meltdown in Cancer Cell Populations.

Large genomes with elevated mutation rates are prone to accumulating deleterious mutations more rapidly than natural selection can purge (Muller's ratchet). As a consequence, it may lead to the extinction of small populations. Relative to most unicellular organisms, cancer cells, with large and nonrecombining genome and high mutation rate, could be particularly susceptible to such "mutational meltdown." However, the most common type of mutation in organismal evolution, namely, deleterious mutation, has received relatively little attention in the cancer biology literature. Here, by monitoring single-cell clones from HeLa cell lines, we characterize deleterious mutations that retard the rate of cell proliferation. The main mutation events are copy number variations (CNVs), which, estimated from fitness data, happen at a rate of 0.29 event per cell division on average. The mean fitness reduction, estimated reaching 18% per mutation, is very high. HeLa cell populations therefore have very substantial genetic load and, at this level, natural population would likely face mutational meltdown. We suspect that HeLa cell populations may avoid extinction only after the population size becomes large enough. Because CNVs are common in most cell lines and tumor tissues, the observations hint at cancer cells' vulnerability, which could be exploited by therapeutic strategies.

In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing.

In this study, we report a novel method for the in situ measurement of autophagy under nutrient starvation using a principle of semiconductor technology. A semiconductor-based field-effect transistor (FET) biosensor enables the direct detection of ionic or molecular charges under biological conditions. In particular, cellular respiration accompanied by the generation of carbon dioxide can be continuously and directly monitored as a change in pH at a cell/sensor interface. When autophagy was induced in HeLa cells on a FET biosensor under nutrient starvation, the surface potential increased more significantly for about 15 h than that for nonstarved cells. This positive shift indicates an increase in the number of hydrogen ions produced from the respiration of starved cells because the sensing surface was previously designed to be sensitive to pH variation. Therefore, we have found that cellular respiration is more activated by autophagy under nutrient starvation because the amino acids that decomposed from proteins in autophagic cells would have been rapidly spent in cellular respiration.

Morphology and dynamics of tumor cell colonies propagating in epidermal growth factor supplemented media.

The epidermal growth factor (EGF) plays a key role in physiological and pathological processes. This work reports on the influence of EGF concentration (c EGF) on the modulation of individual cell phenotype and cell colony kinetics with the aim of perturbing the colony front roughness fluctuations. For this purpose, HeLa cell colonies that remain confluent along the whole expansion process with initial quasi-radial geometry and different initial cell populations, as well as colonies with initial quasi-linear geometry and large cell population, are employed. Cell size and morphology as well as its adhesive characteristics depend on c EGF. Quasi-radial colonies (QRC) expansion kinetics in EGF-containing medium exhibits a complex behavior. Namely, at the first stages of growth, the average QRC radius evolution can be described by a t 1/2 diffusion term coupled with exponential growth kinetics up to a critical time, and afterwards a growth regime approaching constant velocity. The extension of each regime depends on c EGF and colony history. In the presence of EGF, the initial expansion of quasi-linear colonies (QLCs) also exhibits morphological changes at both the cell and the colony levels. In these cases, the cell density at the colony border region becomes smaller than in the absence of EGF and consequently, the extension of the effective rim where cell duplication and motility contribute to the colony expansion increases. QLC front displacement velocity increases with c EGF up to a maximum value in the 2-10 ng ml-1 range. Individual cell velocity is increased by EGF, and an enhancement in both the persistence and the ballistic characteristics of cell trajectories can be distinguished. For an intermediate c EGF, collective cell displacements contribute to the roughening of the colony contours. This global dynamics becomes compatible with the standard Kardar-Parisi-Zhang growth model, although a faster colony roughness saturation in EGF-containing medium than in the control medium is observed.

Carnosine Inhibits the Proliferation of Human Cervical Gland Carcinoma Cells Through Inhibiting Both Mitochondrial Bioenergetics and Glycolysis Pathways and Retarding Cell Cycle Progression.

Carnosine has been demonstrated to play an antitumorigenic role in certain types of cancer. However, its underlying mechanism is unclear. In this study, the roles of carnosine in cell proliferation and its underlying mechanism were investigated in the cultured human cervical gland carcinoma cells HeLa and cervical squamous carcinoma cells SiHa. The results showed that carnosine exerted a significant inhibitory effect on the proliferation of HeLa cells, whereas its inhibitory action on the proliferation of SiHa cells was much weaker. Carnosine decreased the ATP content through inhibiting both mitochondrial respiration and glycolysis pathways in cultured HeLa cells but not SiHa cells. Carnosine reduced the activities of isocitrate dehydrogenase and malate dehydrogenase in TCA (tricarboxylic acid) cycle and the activities of mitochondrial electron transport chain complex I, II, III, and IV in HeLa cells but not SiHa cells. Carnosine also decreased the mRNA and protein expression levels of ClpP, which plays a key role in maintaining the mitochondrial function in HeLa cells. In addition, carnosine induced G1 arrest by inhibiting the G1-S phase transition in both HeLa and SiHa cells. Taken together, these findings suggest that carnosine has a strong inhibitory action on the proliferation of human cervical gland carcinoma cells rather than cervical squamous carcinoma cells. Mitochondrial bioenergetics and glycolysis pathways and cell cycle may be involved in the carnosine action on the cell proliferation in cultured human cervical gland carcinoma cells HeLa.

Magneto-actuated cell apoptosis by biaxial pulsed magnetic field.

We report on a highly efficient magneto-actuated cancer cell apoptosis method using a biaxial pulsed magnetic field configuration, which maximizes the induced magnetic torque. The light transmissivity dynamics show that the biaxial magnetic field configuration can actuate the magnetic nanoparticles with higher responsiveness over a wide range of frequencies as compared to uniaxial field configurations. Its efficacy was demonstrated in in vitro cell destruction experiments with a greater reduction in cell viability. Magnetic nanoparticles with high aspect ratios were also found to form a triple vortex magnetization at remanence which increases its low field susceptibility. This translates to a larger magneto-mechanical actuated force at low fields and 12% higher efficacy in cell death as compared to low aspect ratio nanoparticles.

[Killing effect and its mechanism of low-temperature plasma on different human cancer cell lines].

Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

miR-183 Inhibits UV-Induced DNA Damage Repair in Human Trabecular Meshwork Cells by Targeting of KIAA0101.

PURPOSE: The purpose of this study was to investigate the mechanisms by which miR-183 may contribute to the phenotypic alterations associated with stress-induced senescence of human trabecular meshwork (HTM) cells. METHODS: Changes in gene expression induced by miR-183 in HTM cells were evaluated by gene array analysis, confirmed by quantitative-PCR (Q-PCR), and analyzed by MetaCore pathway analysis. Effects of miR-183 on cell proliferation were assessed by incorporation of bromodeoxyuridine incorporation, and DNA damage by CometAssay after ultraviolet (UV) irradiation in primary HTM cells, and confirmed in human diploid fibroblasts (HDF) and HeLa cells. A plasmid expressing KIAA0101 without its 3'-untranslated region (3'-UTR) was cotransfected with miR-183 to evaluate the role of KIAA0101 on the effects induced by miR-183. RESULTS: miR-183 affected the expression of multiple genes involved in cell cycle regulation and DNA damage response in HTM cells. Forced expression of miR-183 in HTM and HDF resulted in a significant decrease in proliferation in primary HTM and HDF cells but not in HeLa cells. In all cell types tested, overexpression of miR-183 resulted in increased DNA damage under UV irradiation. Expression of KIAA0101 lacking the 3'-UTR region partially prevented the effects of miR-183 on cell proliferation and completely reversed the effects on UV-induced DNA damage. CONCLUSIONS: Our results suggest that the observed up-regulation of miR-183 after stress-induced senescence in HTM cells may contribute to reinforce cellular senescence by inhibiting cell cycle progression through multiple gene targets and limiting the DNA repair mechanisms through inhibition of KIAA0101.

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules.

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset­when the histone mark H2AT120 is dephosphorylated­INCENP and Aurora B switch from centromere to microtubule localization.

Surface glycosaminoglycans protect eukaryotic cells against membrane-driven peptide bacteriocins.

Enzymatic elimination of surface glycosaminoglycans or inhibition of their sulfation provokes sensitizing of HT-29 and HeLa cells toward the peptide bacteriocins nisin A, plantaricin C, and pediocin PA-1/AcH. The effect can be partially reversed by heparin, which also lowers the susceptibility of Lactococcus lactis to nisin A. These data indicate that the negative charge of the glycosaminoglycan sulfate residues binds the positively charged bacteriocins, thus protecting eukaryotic cells from plasma membrane damage.

Automating ChIP-seq experiments to generate epigenetic profiles on 10,000 HeLa cells.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

The effects of the natural enzyme, Pectinex Ultra SP-L, on human cell cultures and bacterial biofilms in vitro.

BACKGROUND: Pectinex Ultra SP-L (Pectinex) is a microbial-derived enzyme that is used in the food industry and that has been shown to inhibit bacterial biofilms. It has been suggested that Pectinex may be useful in the management of biofilm-related bacterial infections and therefore warrants further investigation in this regard. The aim of this study was to investigate the cytotoxicity of Pectinex on cervical adenocarcinoma cells (HeLa), lymphocytes and neutrophils. Cell viability and morphology were assessed using an in vitro spectrophotometric MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay and polarization-optical transmitted light differential interference contrast microscopy. This study also investigated the antibacterial and antibiofilm actions of Pectinex, alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined using p-iodo-nitrotetrazolium violet staining of bacterial cultures and regrowth of subcultures. Biofilm biomass and cell viability were quantified spectrophotometrically after staining with crystal violet and MTT. RESULTS: The IC50 (±SEM) of Pectinex was 193.9 (±22.2) PGU/ml for HeLa cells, 383.4 (±81.5) and 629.6 (±62.8) PGU/ml for fMLP-stimulated and non-stimulated lymphocytes respectively, and 245.9 (±9.4) and 529.7 (±40.7) PGU/ml for fMLP-stimulated and non-stimulated neutrophils, respectively. Induced morphological features characteristic of apoptosis and necrosis included cell membrane blebs and vacuolization in HeLa cells, clumping in lymphocytes, as well as shrunken rounded cells, apoptotic bodies and debris in all cultures. Pectinex (7.42 - 950 PGU/ml-1) was not bactericidal. In clinical cultures of Staphylococcus aureus, co-administration of Pectinex was associated with a 28.0% increase in both the MIC and MBC of amoxicillin-clavulanate. In clinical cultures of P. aeruginosa, there was an 89.0% and 92.8% increase in the MIC and MBC of ciprofloxacin, respectively. Pectinex ≤ 118.75 PGU/ml-1 and incubation periods ≥ 6 h were associated with increased biomass and cell viability in S. aureus or P. aeruginosa biofilms. CONCLUSIONS: Pectinex appeared to antagonize the antibacterial effects of amoxicillin-clavulanate and ciprofloxacin and furthermore demonstrated significant cytotoxicity. It was therefore deemed unsuitable for the management of either planktonic or biofilm phenotypes of S. aureus or P. aeruginosa.

AMP-activated protein kinase is required for cell survival and growth in HeLa-S3 cells in vivo.

Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

Effects of culture media on the susceptibility of cells to apoptotic cell death.

Whether responses of cells to extracellular environments affect the induction of apoptotic cell death is poorly understood. The current study aimed to unravel the different effects of culture media employed in vitro as extracellular environments on the susceptibility of cells to apoptosis. We found that apoptosis is stimulated to the higher levels by culturing human HeLa cells in Opti-MEM with unknown components, a medium that is specifically used for transfections, than by culturing cells in Dulbecco's modified Eagle's medium, a medium that is generally used for maintenance of cells. We showed that apoptosis is suppressed partially by culturing cells in heat-treated Opti-MEM, implicating a heat-sensitive component(s) in stimulating the apoptotic response of cells. Thus, different extracellular environments may contribute to different responses of cells to apoptosis, and this should be considered to evaluate the incidences of apoptotic cell death and could be applied to develop an efficient treatment for curing diseases such as cancer.

High-throughput measurement of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.