Harvard University

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

This movie and the protocol are intended to help learning nuclear transfer.

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system’s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Using multimodal sensors is a promising way to understand the role of social interactions in educational settings. This paper describes a methodology for capturing joint visual attention from colocated dyads using mobile eye-trackers.

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Chronic stroke patients' insured rehabilitation is generally time limited. Imaging-based study of brain activity from walking-related motor tasks can lead to establishing biomarkers to measure improved outcomes and justify extending tailored therapy. A novel, magnetic resonance-compatible, variable-resistance foot motion device and a protocol for use during functional magnetic resonance imaging are presented.

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.