• Volume 12, 2022
  • Volume 11, 2021
  • Volume 10, 2020
  • Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011
Image Credit: BioProtoc.4295

Past Issues in 2022

Volume: 12 Issue: 2

Biological Engineering

Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1104, Q&A: 0

Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides

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Cancer Biology

An Alternative Technique for Monitoring the Live Interaction of Monocytes and Tumor Cells with Nanoparticles in the Mouse Lung Authors:  Fernanda Ramos-Gomes, Nathalia Ferreira, Frauke Alves and M. Andrea Markus, date: 01/20/2022, view: 1162, Q&A: 0

Nanomaterials are increasingly used for the diagnosis and treatment of cancer, including lung cancer. For the clinical translation of nano-based theranostics, it is vital to detect and monitor their accumulation in the tumor, as well as their interaction with tumor, immune cells, and the tumor microenvironment (TME). While high resolution

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Preparation and Cultivation of Colonic and Small Intestinal Murine Organoids Including Analysis of Gene Expression and Organoid Viability Authors:  Luisa Klemke, Joshua P. Blume, Tiago De Oliveira and Ramona Schulz-Heddergott, date: 01/20/2022, view: 1913, Q&A: 0

Organoids are complex three-dimensional structures, which contain different cell types and help to overcome many limitations of conventional 2D cell culture techniques. Here, we present a protocol for the cultivation of murine matched-pairs of small intestinal and colonic epithelial organoids, and colonic tumor organoids derived from the chemical

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Developmental Biology

Combination of Immunofluorescence and Quantitative Fluorescence In-situ Hybridization for Analysing Differential Gene Expression in the Niche Cells of the Drosophila Lymph Gland Authors:  Parvathy Ramesh, Sushmit Ghosh and Lolitika Mandal, date: 01/20/2022, view: 1183, Q&A: 0

The Drosophila larval haematopoietic organ or lymph gland consists of multiple cell types arranged in zones. The smallest stem cell compartment consists of 40-45 cells that constitute the haematopoietic niche. In order to analyse the haematopoietic niche, it needs to be labelled with a specific antibody to differentiate it from the other cell

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Drug Discovery

Rapid in vitro and in vivo Evaluation of Antimicrobial Formulations Using Bioluminescent Pathogenic Bacteria Authors:  Artur Schmidtchen and Manoj Puthia, date: 01/20/2022, view: 1570, Q&A: 0

Basic and translational research needs rapid methods to test antimicrobial formulations. Bioluminescent bacteria and advanced imaging systems capable of acquiring bioluminescence enable us to quickly and longitudinally evaluate the efficacy of antimicrobials. Conventional approaches, such as radial diffusion and viable count assays, are

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Immunology

A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein Authors:  Juliana Conkright-Fincham, Chieri Tomomori-Sato, Rich McGhee, Ella M. Leslie, Carolyn J. Beucher, Lauren E. Weems, Shigeo Sato, William B. Redwine, Kyle J. Weaver, Brandon D. Miller, Kym M. Delventhal, John J. Kary, Andrew B. Koebbe, Alexander Dean, Jessica L. Witt, Laura M. Remy, Tari J. Parmely, Chongbei Zhao, Yan Wang, Joan W. Conaway and Jay R. Unruh, date: 01/20/2022, view: 1253, Q&A: 0

The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative

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Microbiology

Quantification of Bacterial Loads in Caenorhabditis elegans Authors:  Alyssa C. Walker, Rohan Bhargava, Alfonso S. Vaziriyan-Sani, Amanda S. Brust and Daniel M. Czyz, date: 01/20/2022, view: 1269, Q&A: 0

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds.

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High-throughput Growth Measurements of Yeast Exposed to Visible Light Authors:  Katarina Logg, Mikael Andersson, Anders Blomberg and Mikael Molin, date: 01/20/2022, view: 851, Q&A: 0

Light is a double-edged sword: it is essential for life on the planet but also causes cellular damage and death. Consequently, organisms have evolved systems not only for harvesting and converting light energy into chemical energy but also for countering its toxic effects. Despite the omnipresence and importance of such light-dependent effects,

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Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1000, Q&A: 0

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly,

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Molecular Biology

ATAC Sequencing Protocol For Cryopreserved Mammalian Cells Authors:  Juan Manuel Caravaca, Monika Mehta, Sujatha Gowda and Bao Tran, date: 01/20/2022, view: 1380, Q&A: 0

ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful method to evaluate chromatin accessibility and nucleosome positioning at a genome-wide scale. This assay uses a hyperactive Tn5 transposase, to simultaneously cut open chromatin and insert adapter sequences. After sequencing, the reads generated

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Neuroscience

Trichloroacetic Acid Fixation and Antibody Staining of Zebrafish Larvae Authors:  E. Anne Martin, Sundas Ijaz, Alberto E. Pereda and Adam C. Miller, date: 01/20/2022, view: 921, Q&A: 0

Larval zebrafish have been established as an excellent model for examining vertebrate biology, with many researchers using the system for neuroscience. Controlling a fast escape response of the fish, the Mauthner cells and their associated network are an attractive model, given their experimental accessibility and fast development, driving

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Reconstitution of Membrane-associated Components of a G-protein Signaling Pathway on Membrane-coated Nanoparticles (Lipobeads) Authors:  Michael J. Irwin, Xin Wang and Rick H. Cote, date: 01/20/2022, view: 752, Q&A: 0

G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its

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Plant Science

Fractionation and Extraction of Crude Nuclear Proteins From Arabidopsis Seedlings Authors:  Jiajia Zhao, Jinsong Bao and Feifei Xu, date: 01/20/2022, view: 1065, Q&A: 0

The plant nucleus is an important subcellular organelle that contains the genome, ribosomal RNA, and regulatory proteins, and performs a central role in the functioning and metabolism of the cell. Fractionation of intact nuclei is a crucial process to elucidate the function of nuclear proteins. Here, we present a simple method for the

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Rhizoctonia solani Infection Assay of Young Sugar Beet and Arabidopsis plantlets Authors:  Fredrik Dölfors, Louise Holmquist, Georgios Tzelepis and Christina Dixelius, date: 01/20/2022, view: 1295, Q&A: 0

Rhizoctonia solani is a soil-borne fungus, which rarely produces any spores in culture. Hence, all inoculation procedures are based on mycelia, often as a coat on cereal kernels, placed in close vicinity to the plant to be infected. In this protocol, an inoculation method is described where the fungus is first allowed to infest a perlite-maize

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Stem Cell

From 3D to 2D: Harmonization of Protocols for Two-dimensional Cultures on Cell Culture Inserts of Intestinal Organoids from Various Species Authors:  David Warschkau, Estefanía Delgado-Betancourt, David Holthaus, Antonia Müller, Gudrun Kliem, Susanne M. Krug, Joerg-Dieter Schulzke, Toni Aebischer, Christian Klotz and Frank Seeber, date: 01/20/2022, view: 1134, Q&A: 0

In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two-dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological

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Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes Authors:  Mohamed Mohamed Haroon, Praveen Kumar Vemula and Dasaradhi Palakodeti, date: 01/20/2022, view: 919, Q&A: 0

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and

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