Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease‐related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2‐methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well‐defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:220–225, 2015     

Micropatterning and Alignment of Skeletal Muscle Myoblasts Using Microflowed Plasma Process

ObjectivesThe primary objective of the study was to optimize micropatterning environments using the microchannel flowed plasma process for controlling the orientation and behaviour of skeletal muscle cells. We have studied the cellular patterning and alignment of skeletal myoblast cells on the various micropattern widths developed on glass substrates.Materials and MethodsIn this method, we have utilized the microchannel flowed plasma process to create micropatterned self-assembled monolayers of octadecyltrichlorosilane and 3-aminopropyltrichlorosilane for creating cell adhesive widths of 20, 200 and 1000 microns on the glass substrates. The micropatterned substrates were characterized by using fluorescein 5(6)-isothiocyanate. Thereafter, the substrates were used to culture and pattern C2C12 and primary rat skeletal muscle cells. Further, we have studied the spatiotemporal variation in the orientation of the cells by using bright field and fluorescence microscopy. The microscopic images were analysed by using orientation order parameter and orientation distribution analysis.ResultsFITC based characterization of micropatterns reveals that the adopted process for micropatterning can effectively create cell adhesive widths with dimensions comparable to the diameter of myofiber. Microscopic observations and the orientation order parameter analysis reveal the precise alignment and specific orientation of myoblasts along the designated cell adhesive widths that closely mimics the physiological scenario. Both the cells showed immediate alignment within smaller cell adhesive widths of 20 and 200 μm. Actin cytoskeletal staining and its orientation distribution analysis of micropattrned C2C12 cells emphasises the influence of micropatterned environment on cytoskeletal actin orientation.ConclusionThis study corroborates the alignment of the myoblasts using surface cues facilitated by changing surface chemistry of the glass substrates. The study promotes the application of a simple micropatterning technique as a useful tool to regulate the orientation and behaviour of skeletal muscle cells. Also, the study emphasizes the role of spatial topography created by surface modification and its effect on cell adhesion and communication of alignment information across the micropatterns. The microchannel flowed plasma process could be applied to selectively pattern different adherent cell types, which could prove to be a useful platform for the exploration of various cellular processes.     

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.     

Synergistic NGF/B27 gradients position synapses heterogeneously in 3D micropatterned neural cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses.     

Microcontact Peeling as a New Method for Cell Micropatterning

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.     

Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues.     

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.     

Self-adhesive microculture system for extended live cell imaging

Abstract

Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.     

Argon-plasma-induced ultrathin thermal grafting of thermoresponsive pNIPAm coating for contractile patterned human SMC sheet engineering

A new method for ultrathin grafting of pNIPAm on PDMS surfaces is introduced that employs plasma activation of the surface followed by thermal polymerization. This method is optimized for human primary SMC attachment and subsequent intact cell sheet detachment by lowering the temperature. The contractile gene expression of the cells showed that the contractile phenotype of the SMCs which is induced by aligning the cells through micropatterning is more preserved after thermoresponsive cell sheet detachment in contrast with enzymatic detachment. Given its simplicity and low cost, this thermoresponsive grafting method can be utilized for engineering patterned cell sheets for future bottom-up tissue engineering techniques.     

A Fast and Accessible Methodology for Micro-Patterning Cells on Standard Culture Substrates Using Parafilm? Inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm™, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm™ insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm™ insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.     

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.     

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.     

Influence of Micropatterning on Human Periodontal Ligament Cells’ Behavior

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells’ peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells’ stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

User-friendly cell patterning methods using a polydimethylsiloxane mold with microchannels

Techniques for partitioning cell adhesion are useful tools in biological and medical experiments. However, conventional cell patterning methods require special apparatus, special materials or high-level skills. Therefore, we have developed a new cell patterning methodology which can be easily carried out in biological laboratories. Non-cell adhesive material including hydrogel or gas patterns to restrict cell adhesion on a culture dish or glass substrates can be constructed by exploiting a polydimethylsiloxane (PDMS) mold with microchannels. The PDMS molds suck non-adhesive materials into microchannels from the inlet of the microchannels and the materials are immobilized onto the substrates with a desired pattern. High resolution under a few micrometers and long-term stability can be realized. This method has been used for analysis of stem cells, muscle cells, neuron development and other cells in collaboration with many biological researchers. Several examples to use this technique are introduced in this review.     

On-chip cell culture on a microarray of extracellular matrix with surface modification of poly(dimethylsiloxane)

Microfluidic cell culture chips allow to perform assays of small-volume samples rapidly and reproducibly. Most of these chips are made of poly(dimethylsiloxane) (PDMS), which is a flexible, durable, transparent and inexpensive polymer that can be easily applied to fabrication of microstructures by photolithography and replica molding. However, not many cells are able to grow on unmodified PDMS because the cells need appropriate scaffolds on the surface. Here we report surface modification of a PDMS substrate with a microarray of extracellular matrix (ECM) for on-chip cell culture. The ECM proteins collagen and fibronectin were covalently immobilized on an 8 x 8 microarray format by micropatterned UV-induced graft polymerization through a photomask and dehydration-condensation reaction through a microfabricated stencil. Identical spots of ECMs were successfully formed and the geometry of the spots accurately corresponded to the micropattern of the photomask and stencil. We demonstrate the culture of CHO-K1 cells on the ECM microarray chip. Cells proliferated on the fibronectin spots during the 2-day culture.     

A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm™ inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm?, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm? insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm? insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.     

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth

Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.     

Microengineering neocartilage scaffolds

Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.     

A simple technique for quantifying apoptosis in 96-well plates

Background  

Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.