Methods and Protocols — Open Access Journal

The prevalence of non-alcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), is increasing, and as such its contribution to the development of hepatocellular carcinoma is also rising. NAFLD has been shown to influence the immune tumor microenvironment. Therefore, development of pre-clinical mouse models in the context of NAFLD are increasingly important. Here, we describe a mouse model designed to recapitulate the findings of NAFLD followed by rapid induction of orthotopic liver tumors with intrahepatic tumor injection. Additionally, we utilized bioluminescent imaging to monitor tumor growth and response to therapy. The development of one dominant tumor nodule allows precise separation of tumor and liver tissue. This is useful for immunotherapy studies as mononuclear cells from the tumor and the surrounding liver tissue can be analyzed separately. Full article

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting. Full article

The chick chorioallantoic membrane (CAM) is an extra-embryonic membrane, comprised of a high density of blood and lymphatic vessels. CAM has a dense capillary network and is commonly used to study in vivo angiogenesis and anti-angiogenesis in response to potential biomolecules and drugs. Most of the earlier reported CAM assays described the in-ovo method—where the viability of the embryo is higher, but accessibility to the CAM is limited. Ex-ovo CAM methods were previously described that employed shell-less cultures of chick embryos, but the low viability of embryos reduced the overall robustness of the angiogenesis assays. We described a method (named as cup-CAM method) which is more economical, has better accessibility and has significantly improved the viability of the embryo till advanced developmental stages. We could perform this simple yet useful experimentation with the common tools available in the laboratory. We successfully used the cup-CAM method for showing the paracrine effects of conditioned media from tumor cells, on the angiogenesis. This method can be used to assay the angiogenic potential of a drug or protein and to observe the embryonic development of the chick embryo and other related scientific applications. Full article

Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A₂) and the read-through or minor coat protein (A₁), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A₁ protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A₁ is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A₁ protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A₁ proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses. Full article

Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day. Full article

CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation. Full article

Most natural DNA and RNA are devoid of long trinucleotide (TN) sequences that lack one specific nucleotide (missing nucleotide (MN)). Here we developed a novel method that is based on rolling circle amplification (RCA), in which the TN-information of short TN stretches is sequence-specifically recognized, transferred, extended, amplified and detected by padlock probes that consist entirely of nucleotides complementary to the three nucleotides present in the target sequence (complementary TN-information). Upon specific head-to-tail annealing and ligation to the TN-target sequence, these padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by trinucleotide rolling circle amplification (TN-RCA). Since during TN-RCA the MN (as dNTP) is not added, background amplification is minimized with endogenous RNA/DNA (which mostly would require all four dNTP). Therefore, various labelled dNTP can be added to the TN-RCA reaction that enables the separation, isolation and detection of the amplified single-stranded DNA (ssDNA). Here the TN-RCA method is exemplified with RNA/DNA from Zika virus and from human papilloma virus (HPV). TN-RCA is a novel isothermal amplification technique that can be used for sensitive sequence-specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches with low background in short time. Full article

Osteoblast/osteocyte cultures continue to emerge as essential tools for bone biology research in vitro. The change in cell number is an important parameter to be considered for investigating osteogenic differentiation. However, there is no reliable method for quantifying absolute cell count in differentiating osteoblast/osteocyte cultures because of their strongly adherent, multi-layered, super-confluent nature, and their accumulated extracellular matrix production which progressively mineralises in vitro. We developed a practical, simple and cost-effective method based on the fluorometric quantification of a nucleic dye, GelRed™, to enumerate cell number in osteoblast/osteocyte cultures. This method may also be suitable for quantifying cell numbers on other mammalian adherent cell types. Full article

Coomassie brilliant blue (CBB) dyes have been commonly used for the staining of protein bands in polyacrylamide gel electrophoresis (PAGE) gels. However, the staining and destaining of CBB dyes are time-consuming, and the use of methanol is hazardous to one’s health. I introduce a rapid electrophoretic destaining method using a semi-dry transfer unit and a high current power supply. In this method, ethanol was used instead of the hazardous methanol. Most of the protein bands became visible in 30 min. After a secondary destaining step, residual CBB was completely destained. The detection limit for a tested protein (5 ng) was higher than that of the conventional method. Therefore, this method is superior in its speed, safety, low cost, and sensitivity. Full article

In vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. However, these experiments must be complemented with in vitro and ex vivo experiments. Here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy. Full article

India has recently introduced a rotavirus vaccine under a universal immunization program. There is limited information on intussusception, an adverse event, following immunization in children from India. We are conducting sentinel surveillance for intussusception in children aged under two years at 19 hospitals. The sentinel sites’ selection followed a multistage process. The surveillance combines retrospective surveillance for 69 months and prospective surveillance for 18 months. The suspected intussusception cases shall be reviewed for capturing confirmed cases and detailed data collection and classification according to Brighton Collaboration criteria. Data shall be analysed to describe epidemiology, trends, regional and seasonal variations, clinical profiles, management modalities, and outcomes of intussusception. The combination of prospective and retrospective surveillance shall be informative about the trend of intussusception over the last seven years in India. At four sites where rotavirus vaccines have been introduced, the change in intussusception trends shall be documented. The potential association with rotavirus vaccines and other vaccines shall be assessed using case-control and self-controlled case series methodology. Results are forthcoming. The results shall support the national vaccine safety surveillance effort by providing baseline estimates of intussusception for continued monitoring. The surveillance protocol and site selection processes shall inform similar vaccine-safety surveillance in India and other developing countries. Full article

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer’s disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples. Full article

A new method for the measurement of accurate masses using direct infusion in an electrospray-triple quadrupole mass spectrometer is presented and compared to the traditional method using high-resolution mass spectrometry. The proposed method uses internal calibrants and post-acquisition calibration of the mass spectrum signal using the MassWorks software to determine accurate masses. Then, based on parameters such as elemental composition, number of double bond equivalents, and type of ion (even- or odd-electron), etc., a list of potential molecular formula candidates are generated and ranked according to spectral accuracy, (i.e., similarity between the calibrated profile and theoretical isotopic patterns). Experiments using six diverse synthesis products showed that mass accuracy in the Quattro Premier triple quadrupole mass spectrometer (QqQMS) was ≤9.2 mDa and spectral accuracy was ≥90.6%. According to both mass accuracy tolerance (±10 mDa) and spectral accuracy, the correct molecular formula was ranked in the top seven compounds out of up to 32 potential candidates. When considering the context of the synthesis reaction, only one formula was possible. In summary, results showed that the measurement of spectral accuracy in a low-resolution instrument such as the triple quadrupole was strongly dependent on the signal intensity and the presence of interfering peaks in the profile mass range window. This study suggests that use of triple quadrupole mass spectrometry followed by post-acquisition calibration can be an economical and robust approach compared to the traditional method using high-resolution mass spectrometers for the measurement of accurate masses in routine applications using small organic molecules at microgram-per-litter concentrations in relatively clean matrices. Full article

Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed. Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. Additionally, it will provide a starting point for the development of pseudotype neutralization assays using pseudotypes bearing other viral envelope proteins. Full article

Positron Emission Tomography scan images are extensively used in radiotherapy planning, clinical diagnosis, assessment of growth and treatment of a tumor. These all rely on fidelity and speed of detection and delineation algorithm. Despite intensive research, segmentation has remained a challenging problem due to the diverse image content, resolution, shape, and noise. This paper presents a fast positron emission tomography tumor segmentation method using superpixels. Principal component analysis is applied on the superpixels and their average value. The distance vector of each superpixel from the average is computed in the principal components coordinate system. Finally, k-means clustering is applied on the distance vector to recognize tumor and non-tumor superpixels. The proposed approach is implemented in MATLAB 2016A, and promising accuracy with execution time of 2.35 ± 0.26 s is achieved. Fast execution time is achieved since the number of superpixels, and the size of distance vector on which clustering was done are low compared to the number of pixels in the image. Full article

The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus). Full article

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2–4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection. Full article

A single solution protocol has been widely used for the fluorimetric determination of H₂O₂ in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin and H₂O₂ in the sample and the subsequent internal additions, and the measurements of the fluorescence are all carried out at a single pH in a fluorometer cell. It is found that this protocol is prone to four sources of possible error. The variability in the reaction stoichiometry between scopoletin and H₂O₂ in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H₂O₂, the photobleaching of scopoletin, and the de-activation of HRP. These possible sources of error can be circumvented in a two-stage protocol in which the reaction between H₂O₂ and scopoletin is carried out immediately upon sampling at a pH of 7, and the measurement of the fluorescence is carried out later on at a pH of 9. It should be the protocol of choice. Furthermore, in the two-stage protocol, after the initial reaction between H₂O₂ and scopoletin, the sample may be stored at room temperature for six days and at 4 °C for at least a month before its fluorescence is measured. This option can significantly reduce the logistics in the field. Full article

While many methods exist to quantitatively determine protein kinase activities, 32P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for phosphorylation by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation sensors, can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio. Full article