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Past Issue in 2019
Volume: 9, Issue: 21
Cancer Biology
Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy
Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, EM, and CLEM. However, tagging and tracking cellular proteins across size scales is restricted by the scarcity of methods for attaching appropriate reporter chemistries to target proteins. Namely, there are few genetic tags compatible with EM. To overcome these issues we developed Versatile Interacting Peptide (VIP) tags, genetically-encoded peptide tags that can be used to image proteins by fluorescence and EM. VIPER, a VIP tag, can be used to label cellular proteins with bright, photo-stable fluorophores for FM or electron-dense nanoparticles for EM. In this Bio-Protocol, we provide an instructional guide for implementing VIPER for imaging a cell-surface receptor by CLEM. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).
F-actin Bundle Sedimentation Assay
Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and aligning them into actin bundles. Here we utilize dynamin as an example and present a simple protocol to analyze the actin bundling activity in vitro. This protocol details the method for F-actin reconstitution as well as quantitative and qualitative analyses for actin bundling activity of dynamins. Measurement of the actin bundling activity of other actin-binding proteins may also be applied to this protocol with appropriate adjustments depending on the protein of interest.
Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy
Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other (“probe peptide”) delivering a reporter compatible with imaging. Heterodimer formation is rapid and specific, allowing proteins to be selectively labeled for live-cell and fixed-cell imaging. In this Bio-Protocol, we include a detailed guide for implementing the VIPER technology for imaging receptors on live cells and intracellular targets in fixed cells. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).
Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins
Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag (Doh et al., 2018), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).
Cell Biology
High Throughput Traction Force Microscopy for Multicellular Islands on Combinatorial Microarrays
The composition and mechanical properties of the cellular microenvironment along with the resulting distribution of cellular devolved forces can affect cellular function and behavior. Traction Force Microscopy (TFM) provides a method to measure the forces applied to a surface by adherent cells. Numerous TFM systems have been described in literature. Broadly, these involve culturing cells on a flexible substrate with embedded fluorescent markers which are imaged before and after relaxion of cell forces. From these images, a displacement field is calculated, and from the displacement field, a traction field. Here we describe a TFM system using polyacrylamide substrates and a microarray spotter to fabricate arrays of multicellular islands on various combinations of extra cellular matrix (ECM) proteins or other biomolecules. A microscope with an automated stage is used to image each of the cellular islands before and after lysing cells with a detergent. These images are analyzed in a semi-automated fashion using a series of MATLAB scripts which produce the displacement and traction fields, and summary data. By combining microarrays with a semi-automated implementation of TFM analysis, this protocol enables evaluation of the impact of substrate stiffness, matrix composition, and tissue geometry on cellular mechanical behavior in high throughput.
Developmental Biology
Isolation and Culture of Murine Hepatic Stellate Cells
Hepatic stellate cells (HSCs), alternatively known as liver pericytes, can differentiate into myofibroblasts and secrete extra-cellular matrix components, thereby promoting wound healing and fibrosis. Studying HSCs can provide insights into the pathological mechanisms governing these processes. HSC isolation methods typically comprise of enzymatic digestion followed by density gradient centrifugation and/or Fluorescent Activated Cell Sorting (FACS) mediated sorting. In this protocol, we describe a step-wise method for HSC isolation that utilizes Pronase and Collagenase for enzymatic tissue dissociation, followed by an Optiprep based density gradient centrifugation. The isolation can be performed using common media and buffers, and without the use of any special equipment for liver perfusion and HSC isolation. The technique yields ex vivo HSCs, suitable for use in assays.
Immunology
Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans
Reagents such as Amplex® Red have been developed for detecting hydrogen peroxide (H2O2) and are used to measure the release of H2O2 from biological samples such as mammalian leukocytes undergoing the oxidative burst. Caenorhabditis elegans is commonly used as a model host in the study of interactions with microbial pathogens and releases reactive oxygen species (ROS) as a component of its defense response. We adapted the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit to measure H2O2 output from live Caenorhabditis elegans exposed to microbial pathogens. The assay differs from other forms of ROS detection in the worm, like dihydrofluorescein dyes and genetically encoded probes such as HyPer, in that it generally detects released, extracellular ROS rather than intracellular ROS, though the distinction between the two is blurred by the fact that certain species of ROS, including H2O2, can cross membranes. The protocol involves feeding C. elegans on a lawn of the pathogen of interest for a period of time. The animals are then rinsed off the plates in buffer and washed to remove any microbes on their cuticle. Finally, the animals in buffer are distributed into 96-well plates and Amplex® Red and horseradish peroxidase (HRP) are added. Any H2O2 released into the buffer by the worms will react with the Amplex® Red reagent in a 1:1 ratio in the presence of HRP to produce the red fluorescent excitation product resorufin that can be measured fluorometrically or spectrophotometrically, and the amount of H2O2 released can be calculated by comparison to a standard curve. The assay is most appropriate for studies focused on released ROS, and its advantages include ease of use, the ability to use small numbers of animals in a plate reader assay in which measurements can be taken either fluorometrically or spectrophotometrically.
Adoptive Transfer of Basophils Enriched from Mouse Spleen
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CD49b is a member of the integrin family, expressed on basophils, natural killer (NK) cells and a subset CD4+ T cells in the spleen. This protocol describes the adoptive transfer of basophil-enriched CD49b+ cells obtained from mouse spleens by magnetic enrichment. This protocol can be used to assess the contribution of basophils or basophil-derived mediators to a certain immune response.
Microbiology
A Mismatch-tolerant RT-LAMP Method for Molecular Diagnosis of Highly Variable Viruses
Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens. However, there are usually numerous variants in one viral pathogen and primers employed in LAMP can hardly match all these variants. The mismatches between the primers and the viral genomes, especially those at the 3′-end of the primers, hinder LAMP reactions, leading to failure of the detection. Here, we present a mismatch-tolerant RT-LAMP protocol, which utilizes the 3′-5′ exonuclease activity of the Q5 high-fidelity DNA polymerase to remove potential mismatched bases at the 3′-end of the primers during LAMP amplification. Using HIV-1 as a proof-of-principle, we showed that this protocol could represent a promising tool for accurate detection of genetically unstable viruses in laboratory, hospital and field.
Purification and HPLC Analysis of Cell Wall Muropeptides from Caulobacter crescentus
The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall including muropeptide composition, glycan strand length, degree of crosslinking, type of crosslinking and other peptidoglycan modifications can be quantified using this approach. Well-established protocols provide us with highly-resolved and quantitatively reproducible chromatographic data, which can be used to investigate bacterial cell wall composition under a variety of environmental or genetic perturbations. The method described here enables the purification of muropeptide samples, their quantification by HPLC, and fraction collection for peak identification by mass spectrometry. Although the methods for peptidoglycan purification and HPLC analysis have been previously published, our method includes important details on how to re-equilibrate the column between runs to allow for automated analysis of multiple samples.
In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values
Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols for the in vitro screening of a natural product-derived compound required for the determination of its antileishmanial potency. For this purpose, the Total Phenolic Fraction (TPF) derived from extra virgin olive oil is evaluated through the in vitro cell culture method against extracellular promastigote and intracellular amastigote Leishmania spp. forms. The aim of this article is to describe a step-by-step procedure that can be easily applied to accurately estimate the 50% inhibitory concentration (IC50), the 50% cytotoxic concentration (CC50) and the selectivity index (SI) via the resazurin reduction assay. These protocols are based on the ability of resazurin (oxidized blue form) to be irreversibly reduced by enzymes in viable cells and generate a red fluorescent resorufin product and can be easily expanded to the investigation of the antimicrobial activity in other microorganisms.
Model of Chemotherapy-associated Mucositis and Oral Opportunistic Infections
Oral mucositis is a common complication of cancer chemotherapy treatment. Because of the lack of relevant oral mucositis experimental models, it is not clear how chemotherapeutic agents injure the oral mucosa and if commensal microorganisms accelerate tissue damage. We developed an organotypic oral mucosa model that mimics cellular responses commonly associated with cytotoxic chemotherapy. The organotypic model consists of multilayer oral epithelial cells growing over a collagen type I matrix, with embedded fibroblasts. Treatment of organotypic constructs with the chemotherapeutic agent, 5-fluorouracil (5-FU), leads to major histopathologic changes resembling mucositis, such as DNA synthesis inhibition, increased apoptosis and cytoplasmic vacuolation. Candida albicans formed mucosal biofilms on these tissues and augmented the inflammatory responses to 5-FU. This model can be used in further mechanistic studies of oral mucositis and associated opportunistic infections.
Molecular Biology
Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine
The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry method for use with an imaging flow cytometer (Amnis FlowSight) for identifying EV released by specific cell types and excreted into the urine or blood using markers characteristic of particular cells in the kidney. Here we present a protocol utilizing the Amnis FlowSight Imaging Flow Cytometer to identify and quantify EV from the urine of patients with essential hypertension and renovascular disease. Notably, EV isolated from cell culture media and plasma can also be analyzed similarly.
Neuroscience
Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM
In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1). We used nickel beads to isolate His-tagged KIN-19 and RHO-1, and thus permitting the isolation of both small and large aggregated or fibrillary forms of the proteins. We characterized their morphology by transmission electron microscopy. We further expressed RFP-tagged proteins and stained them with a fluorescent molecule, thioflavin T, which identifies β-sheet structures, and which is a defining feature of amyloid fibrils. We further applied structured illumination microscopy to determine the level of colocalization between RFP and thioflavin T.
Stem Cell
Differentiation of Mouse Embryonic Stem Cells to Neuronal Cells Using Hanging Droplets and Retinoic Acid
Controlled differentiation of embryonic stem cells is an essential tool in stem cell research. In this protocol, we describe a simple differentiation protocol involving the induction of embryoid body formation in mouse embryonic stem cells (mESC) using hanging droplets, followed by differentiation into a neuronal lineage.