Standard Polystyrene Research Articles

Marine plastics alter the organic matter composition of the air-sea boundary layer, with influences on CO2 exchange: a large-scale analysis method to explore future ocean scenarios

Microplastics are substrates for microbial activity and can influence biomass production. This has potentially important implications in the sea-surface microlayer, the marine boundary layer that controls gas exchange with the atmosphere and where biologically produced organic compounds can accumulate. In the present study, we used six large scale mesocosms to simulate future ocean scenarios of high plastic concentration. Each mesocosm was filled with 3 m3 of seawater from the oligotrophic Sea of Crete, in the Eastern Mediterranean Sea. A known amount of standard polystyrene microbeads of 30 μm diameter was added to three replicate mesocosms, while maintaining the remaining three as plastic-free controls. Over the course of a 12-day experiment, we explored microbial organic matter dynamics in the sea-surface microlayer in the presence and absence of microplastic contamination of the underlying water. Our study shows that microplastics increased both biomass production and enrichment of carbohydrate-like and proteinaceous marine gel compounds in the sea-surface microlayer. Importantly, this resulted in a ∼3 % reduction in the concentration of dissolved CO2 in the underlying water. This reduction was associated to both direct and indirect impacts of microplastic pollution on the uptake of CO2 within the marine carbon cycle, by modifying the biogenic composition of the sea's boundary layer with the atmosphere.

Development of a differential photoacoustic system for the determination of the effective water diffusion and water vapor permeability coefficients in thin films

This paper focused on developing a methodology and metrology using a differential photoacoustic (PA) system to determine the effective water vapor diffusion coefficient (Deff) and the effective permeability coefficient (Π) in thin films as a piece of paper and standard polystyrene for a controlled relative humidity. The methodology proposes a new differential photoacoustic system, including the water reservoir, relative humidity, and temperature detectors. Two cells, reference/sample, were used to obtain the instrumental function to reduce the electronic and environmental noises. A method based on the study of ln[1−(S−S0)/ΔS]=t/τD and the behaviors of R2 as a function of the number of data was proposed to assess the region in which the photoacoustic signal should be processed to determine each effective coefficient. S is the amplitude of the PA signal, S0 is the initial amplitude value, ΔS is the change, t (time), and τD is the water vapor diffusion time. The effective water diffusion coefficient (Deff) for water and polystyrene was 1.90 × 10−11 m2/s and 3.09 × 10−11 m2/s, respectively. The permeability coefficient value for the piece of paper was 4.18 × 10−9 mol kg−1 cm−2 s−1 Pa−1, while for polystyrene, it was 6.80 × 10−9 mol kg−1 cm−2 s−1 Pa−1 for 70% of relative humidity. This methodology can be extended by changing the moisture content on the chamber to obtain the dependence of Deff as a function of relative humidity.

High resolution DLP stereolithography to fabricate biocompatible hydroxyapatite structures that support osteogenesis.

Lithography based additive manufacturing techniques, specifically digital light processing (DLP), are considered innovative manufacturing techniques for orthopaedic implants because of their potential for construction of complex geometries using polymers, metals, and ceramics. Hydroxyapatite (HA) coupons, printed using DLP, were evaluated for biological performance in supporting viability, proliferation, and osteogenic differentiation of the human cell line U2OS and human mesenchymal stem cells (MSCs) up to 35 days in culture to determine feasibility for future use in development of complex scaffold geometries. Contact angle, profilometry, and scanning electron microscopy (SEM) measurements showed the HA coupons to be hydrophilic, porous, and having micro size surface roughness, all within favourable cell culture ranges. The study found no impact of leachable and extractables form the DLP printing process. Cells seeded on coupons exhibited morphologies comparable to conventional tissue culture polystyrene plates. Cell proliferation rates, as determined by direct cell count and the RealTime-GloTM MT Cell Viability Assay, were similar on HA coupons and standard tissue culture polystyrene plates). Osteogenic differentiation of human MSCs on HA coupons was confirmed using alkaline phosphatase, Alizarin Red S and von Kossa staining. The morphology of MSCs cultured in osteogenic medium for 14 to 35 days was similar on HA coupons and tissue culture polystyrene plates, with osteogenic (geometric, cuboidal morphology with dark nodules) and adipogenic (lipid vesicles and deposits) features. We conclude that the DLP process and LithaBone HA400 slurry are biocompatible and are suitable for osteogenic applications. Coupons served as an effective evaluation design in the characterization and visualization of cell responses on DLP printed HA material. Results support the feasibility of future technical development for 3D printing of sophisticated scaffold designs, which can be constructed to meet the mechanical, chemical, and porosity requirements of an artificial bone scaffold.

Identification of Trace Polystyrene Nanoplastics Down to 50 nm by the Hyphenated Method of Filtration and Surface-Enhanced Raman Spectroscopy Based on Silver Nanowire Membranes.

Nanoplastics are emerging pollutants that pose potential threats to the environment and organisms. However, in-depth research on nanoplastics has been hindered by the absence of feasible and reliable analytical methods, particularly for trace nanoplastics. Herein, we propose a hyphenated method involving membrane filtration and surface-enhanced Raman spectroscopy (SERS) to analyze trace nanoplastics in water. In this method, a bifunctional Ag nanowire membrane was employed to enrich nanoplastics and enhance their Raman spectra in situ, which omitted sample transfer and avoided losing smaller nanoplastics. Good retention rates (86.7% for 50 nm and approximately 95.0% for 100-1000 nm) and high sensitivity (down to 10-7 g/L for 50-1000 nm and up to 105 SERS enhancement factor) of standard polystyrene (PS) nanoplastics were achieved using the proposed method. PS nanoplastics with concentrations from 10-1 to 10-7 g/L and sizes ranging from 50 to 1000 nm were successfully detected by Raman mapping. Moreover, PS micro- and nanoplastics in environmental water samples collected from the seafood market were also detected at the μg/L level. Consequently, the proposed method provides more possibilities for analyzing low-concentration nanoplastics in aquatic environments with high enrichment efficiency, minimal sample loss, and high sensitivity.

Accurate 3D morphological computational model reconstruction of suspended cells imaged through stratified media by the precise depth-varying point spread function method.

Accurate three-dimensional (3D) morphological computational models of cells are important in a number of biological studies. This study proposes a precise depth-varying point spread function (PDV-PSF) method for reconstructing 3D computational models of suspended cells from two-dimensional (2D) confocal image stacks. Our approach deblurs the 2D images in horizontal plane and corrects the deformation in vertical direction to overcome the refractive index mismatch problem caused by suspended cells imaging through stratified media. Standard fluorescent polystyrene spheres and Jurkat T-lymphocytes are selected to evaluate the validity and accuracy of this PDV-PSF method. Qualitative and quantitative results demonstrate that our approach has superior performance in 3D morphological computational models reconstruction of suspended cells.

Metal-Catalyzed, Photo-Assisted Selective Transformation of Tertiary Alkylbenzenes and Polystyrenes into Carbonyl Compounds.

Every year, thousands of tons of polystyrene are produced and discarded, filling landfills and polluting the marine environment. Although several degradation alternatives have been proposed, the need for an effective procedure for the chemical recycling of polystyrene still remains. Here, a vanadium‐catalyzed reaction, assisted by visible light, promoted the direct, selective conversion of tertiary alkylbenzenes into acetophenone and other ketone derivatives. Likewise, standard polystyrene samples as well as polystyrenes from insulation and packaging waste could be chemically recycled into acetophenone in a scalable way regardless of their molecular weight, polydispersity, or form. Preliminary mechanistic investigations revealed the participation of singlet oxygen, superoxide, and hydroxyl radical species in this homogenously catalyzed process. Acetophenone could be used as an additive to accelerate the reaction and to increase the yields in some cases.

Towards a fast and generalized microplastic quantification method in soil using terahertz spectroscopy

Extensive investigation of microplastic abundance in soil environment calls for rapid, accurate, efficient and harmonized quantification methods. Development of rapid quantification method requires made-to-measure soil samples with additions of standard polymers. Existing rapid quantification methods ignore the gap between standard polymers in laboratory and household microplastics in soil environment. Here, terahertz (0.6–1.67 thz) and NIR (950–1660 nm) spectroscopy were compared to explore a fast, accurate and potentially generalizable microplastic quantification method in soil. Soil sample was spiked with two standard polymers (polyvinyl chloride (PVC) and polystyrene (PS)) and their additive-containing household microplastics. Two standard sample sets and two household sample sets were prepared in concentrations ranging from 0.5 to 10%. Nine commonly used preprocessing methods and three machine learning algorithms were coupled to develop methods. Models were constructed by training sets from standard sample sets. When models transferred to household samples, prediction error (RMSE) of proposed terahertz method (Wdenosie_PLSR) only increased by 0.4% for PVC and 0.19% for PS, yet that of the NIR method increased by 1.49% and 1.16% respectively. The proposed terahertz method presented a detection limit around 1.12% and the NIR method showed a detection limit around 3.24%. Overall, our results suggest that compared with NIR method, the proposed terahertz method is not only more accurate but also demonstrate stronger generalizability to bridge the gaps between standard PVC/PS polymers and household PVC/PS microplastics. We also propose MMD heatmap for diagnosing spectral preprocessing methods to further improve method efficiency.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices.

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

Single-Shot Common-Path Off-Axis Dual-Wavelength Digital Holographic Microscopy Based on Two-Dimensional Grating Diffraction

We present a single-shot dual-wavelength common-path off-axis digital holographic microscopic (CO-DHM) imaging method based on two-dimensional grating diffraction. This method improves the utilization rate of the interference field under the limited photosensitive size of the camera, and further expands the original camera’s field of view (FOV). In addition, the mode of orthogonal carrier frequencies close to the diagonal direction can optimize the utilization of the camera’s spatial bandwidth. Compared with the traditional dual-wavelength CO-DHM using one-dimensional grating or prism beam splitting, this method effectively avoids the aliasing of high-frequency components of the +1-order spectrum of different wavelengths in the frequency domain. We provide quantitative phase imaging experiments for the full FOV of USAF resolution chart, onion epidermal cells and standard polystyrene beads. The results prove that the system can enlarge the interferometric FOV by nearly 74.0% without changing the imaging parameters, such as magnification and resolution, and can achieve high-precision quantitative phase imaging with only a single hologram.

Dual-wavelength extinction rainbow refractometry for in-situ characterization of colloidal droplets

Considerable interest and demand exists in multi-parameter characterization of colloidal droplets. However, an optical in-situ characterization of such droplets is a particular challenge due to its heterogeneous and multi-scale nature, motivating the investigation of a dual-wavelength extinction rainbow refractometry. An analytical model is proposed, in which dual-wavelength extinction effect of the inclusions on the amplitude of the primary rainbow peak allows measurement of inclusion concentration and particle size. The rainbow pattern itself yields the refractive index and diameter of the host droplet. Validation experiments using deionized monodisperse water droplets containing standard polystyrene nanoparticles with different concentrations (0%, 0.1%, 0.2%, 0.3%) and particle sizes (210 nm, 530 nm) were conducted. Measured refractive indices and droplet diameters agree well with the known values. Measurement errors of inclusion concentration and size were analyzed with and without a prior knowledge of the inclusion size. Measurement limits of this approach are also discussed.

Geometry Optimalization of the Ventilated Insulating Panel

Abstract An EPS ventilated panel, which may be applied as an external insulation to humid walls, is investigated. Dimensions of the air channel sections have been determined using the Ansys software. Afterwards, the drying rate of the walls externally insulated with EPS, mineral wool and EPS ventilated panel has been compared using the WUFI software. The ventilated panel increases the drying rate when compared with the standard polystyrene panel and increases the heat loss through the wall by less than 20%.

A Novel Method for 3D Nanoscale Tracking of 100 nm Polystyrene Particles in Multi-Wavelength Evanescent Fields Microscopy – Absolute Difference Height Verification –

Total internal reflection is an optical imaging technique for nanoparticle tracking and observation employing the scattered light from an evanescent field near the interface or reference surface. Generally, the nanoparticle behavior is the three-dimensional Brownian motion in an aqueous medium. The motion can be traced by an optical microscopy, but it cannot be traced by an electron microscopy technique. In the three-dimensional nanoparticle moving position, the X and Y positions are parallel to the surface, which can be traced by the general microscopy techniques. However, the height position Z of a nanoparticle perpendicular to the surface could not be traced without the longitudinal scanning method. Here, a novel method is proposed to investigate the 3D position of nanoparticles by applying multi-wavelength evanescent fields microscopy, which has a high spatial resolution in the Z-direction without longitudinal scanning. This paper focuses on the verification of measurement in the Z-direction. A piezoelectric actuator was employed to control the nanoparticle displacement in height Z. Standard polystyrene 100 nm particles were randomly adhered on a spherical tip that connected with the piezoelectric actuator. The spherical tip was essentially made from an optical adhesive (n = 1.348) with a refractive index close to the water for decreasing the unnecessary signal from the tip-self during nanoparticle observation in the water. The proposed method could obtain the multi-wavelength scattering lights from the observed nanoparticles by an 8-bit color camera with higher than 50 frames per second recording to investigate the 3D nanoscale tracking. The X and Y positions of nanoparticles were determined by the centroid of the scattering light intensities. The height Z was determined from the logarithm ratios between the detected scattering light intensities of both wavelengths. The measurement repeatability of the absolute difference in height between nanoparticles could be measured less than ±16 nm by using the proposed method. The penetration height measurability range was approximated at 250 nm from the reference surface.

Measuring particle size in ultra-low concentration suspensions by removing the number fluctuation contribution in dynamic light scattering.

In ultra-low concentration suspensions, particle number fluctuations in the scattering volume add a long delay component to the intensity autocorrelation function (ACF) in dynamic light scattering (DLS) measurements. This gives a strong artifact peak in the particle size distribution (PSD) recovered. To improve the accuracy of DLS at ultra-low concentrations, we analyzed the different decay characteristics of particle Brownian motion and particle number fluctuation in the ACF. By differentiating the ACF we were able to identify and separate the number fluctuation term and then analyze the ACF to recover the PSD. The results for simulated DLS data at 151nm and 690nm diameters with average particle numbers of 6, 12, 24 and 48 in the scattering volume at four noise levels show that, compared with the usual DLS data processing method, inversion of the ACF after the separation of the number fluctuation term effectively eliminates the strong artifact peaks, and the relative errors of peak positions and distribution errors are significantly reduced. This was further verified with experimental results from samples of standard polystyrene spheres.

In-situ Detection Method for Microplastics in Water by Polarized Light Scattering

Microplastics (MPs) have become the widespread contaminants, which raises concerns on their ecological hazards. In-situ detection of MP in water bodies is essential for clear assessment of the ecological risks of MPs. The present study proposes a method based on polarized light scattering which measures the polarization parameters of the scattered light at 120° to detect MP in water. This method takes the advantage of in-situ measurement of the individual particles and the experimental setup in principle is used. By use of the measured polarization parameters equipped by machine learning, the standard polystyrene (PS) spheres, natural water sample, and lab-cultured microalgae are explicitly discriminated, and MP with different physical and chemical properties can be differentiated. It can also characterize the weathering of different MP and identify the specific type from multiple types of MP. This study explores the capability of the proposed method to detect the physical and chemical properties, weathering state and concentration of MP in water which promises the future application in water quality sensing and monitoring.

Microfluidics on Standard Petri Dishes for Bioscientists.

Few microfluidic devices are used in biomedical labs, despite the obvious potential; reasons given include the devices are rarely made with cell-friendly materials, and liquids are inaccessibly buried behind solid confining walls. An open microfluidic approach is reviewed in which aqueous circuits with almost any imaginable 2D shape are fabricated in minutes on standard polystyrene Petri dishes by reshaping two liquids (cell-culture media plus an immiscible and bioinert fluorocarbon, FC40). Then, the aqueous phase becomes confined by fluid FC40 walls firmly pinned to the dish by interfacial forces. Such walls can be pierced at any point with pipets and liquids added or removed through them, while flows can be driven actively using external pumps or passively by exploiting local differences in Laplace pressure. As walls are robust, permeable to O2 plus CO2 , and transparent, cells are grown in incubators and monitored microscopically as usual. It is hoped that this simple, accessible, and affordable fluid-shaping technology provides bioscientists with an easy entrée into microfluidics.

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use.

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use cups and kettles) indicate that MP release during daily use can be extremely high. Precisely determining the MP release level is a crucial step to identify and quantify the exposure source and assess/control the corresponding risks stemming from this exposure. Though protocols for measuring MP levels in marine or freshwater has been well developed, the conditions experienced by household plastic products can vary widely. Many plastic products are exposed to frequent high temperatures (up to 100 °C) and are cooled back to room temperature during daily use. It is therefore crucial to develop a sampling protocol that mimics the actual daily-use scenario for each particular product. This study focused on widely used polypropylene-based baby feeding bottles to develop a cost-effective protocol for MP release studies of many plastic products. The protocol developed here enables: 1) prevention of the potential contamination during sampling and detection; 2) realistic implementation of daily-use scenarios and accurate collection of the MPs released from baby feeding bottles based on WHO guidelines; and 3) cost-effective chemical determination and physical topography mapping of MPs released from baby feeding bottles. Based on this protocol, the recovery percentage using standard polystyrene MP (diameter of 2 µm) was 92.4-101.2% while the detected size was around 102.2% of the designed size. The protocol detailed here provides a reliable and cost-effective method for MP sample preparation and detection, which can substantially benefit future studies of MP release from plastic products.

Life cycle assessment during packaging of market-sized seabass and meagre: necessary adaptations toward GHG neutrality.

PurposeFish is a delicate and valuable source of protein, and aquaculture is expected to provide the required amount of fish needed at reasonable prices. Packaging is a vital stage to preserve hygiene, quality and freshness of aquaculture products. The purpose of this study was to identify environmental hotspots in farmed seabass and meagre during the packaging stage and to examine scenarios for reducing environmental impacts, aiming at greenhouse gas neutrality.MethodsThe life cycle assessment method was applied for the first time at three packaging plants of farmed seabass and meagre in western Greece, in order to assess their environmental impacts, taking into account the amount of electricity, expanded polystyrene, and other plastic and packaging materials needed. Moreover, different scenarios were designed involving the energy mix/alternative sources, recycling and reuse of packaging materials, and the replacement of standard polystyrene boxes and wooden pallets, in order to suggest environmentally friendly improvements in the packaging process.Results and discussionConventional operation of seabass and meagre packaging plants requires significant amounts of energy and packaging materials. In fact, electricity, boxes and pallets were documented as the main contributors to most of the environmental impact indicators. Seabass packaging had a slightly lower overall environmental impact compared to meagre, due to its smaller market size. Impact minimization scenarios resulted in various degrees of reduction of environmental burdens in both species; however, near-zero GHG emissions were achieved when energy from photovoltaic panels and recycling/reuse of packaging materials were combined with the use of non-fossil-based boxes and recyclable pallets. Such readily applicable adjustments to the conventional operation of packaging plants would contribute to overall environmental sustainability and to better consumer acceptability of the final product.ConclusionsIn a nutshell, the life cycle assessment method proved to be a valuable tool for assessing the environmental performance of Mediterranean aquaculture packaging processes. Moreover, it served to identify critical factors directly related to the EU GHG neutrality target. Accordingly, environmentally friendly decision-making/strategic management in the farmed fish packaging sector will inevitably rely on alternative energy sources, recycling of packaging materials, and use of recyclable corrugated board boxes or similar non-fossil-based materials.

Deep-Learning-Based Halo-Free White-Light Diffraction Phase Imaging

In white-light diffraction phase imaging, when used with insufficient spatial filtering, phase image exhibits object-dependent artifacts, especially around the edges of the object, referred to the well-known halo effect. Here we present a new deep-learning-based approach for recovering halo-free white-light diffraction phase images. The neural network-based method can accurately and rapidly remove the halo artifacts not relying on any priori knowledge. First, the neural network, namely HFDNN (deep neural network for halo free), is designed. Then, the HFDNN is trained by using pairs of the measured phase images, acquired by white-light diffraction phase imaging system, and the true phase images. After the training, the HFDNN takes a measured phase image as input to rapidly correct the halo artifacts and reconstruct an accurate halo-free phase image. We validate the effectiveness and the robustness of the method by correcting the phase images on various samples, including standard polystyrene beads, living red blood cells and monascus spores and hyphaes. In contrast to the existing halo-free methods, the proposed HFDNN method does not rely on the hardware design or does not need iterative computations, providing a new avenue to all halo-free white-light phase imaging techniques.

Isolation and culture of adult murine cardiac atrial and ventricular fibroblasts and myofibroblasts.

Cardiac fibroblasts play a critical role in extracellular matrix homeostasis, wound healing, and cardiac interstitial fibrosis: the latter being a pathophysiological response to a chronic increase in afterload. Using a standard protocol to isolate cardiac fibroblasts and maintain them in their quiescent phenotype in vitro will enable a better understanding of cardiac fibroblast biology and their role in the response to profibrotic stimuli. Here, we describe an enzymatic method for isolating cardiac fibroblasts. The resulting cells are maintained on either a collagen-coated hydrogel-bound polystyrene (compliant) substrate or standard polystyrene culture dishes (non-compliant) to obtain quiescent fibroblasts and activated fibroblasts (myofibroblasts), respectively. Fibroblasts maintained on a non-compliant substrate developed a myofibroblast phenotype, in which the αSMA immunoreactivity was markedly elevated and incorporated into the stress fibers. In contrast, ventricular and atrial fibroblasts retain their quiescent phenotype for up to 3 passages when maintained on a compliant substrate. Hence, the methodology described herein provides a simple and reproducible way to isolate adult murine atrial and ventricular cardiac fibroblasts from a single animal and, by selecting a substrate with the appropriate compliance, examine the mediators of fibroblast activation or inactivation.