Improve Research Reproducibility A Bio-protocol resource
- Submit a Protocol
- Receive Our Alerts
- EN
- Protocols
- Articles and Issues
- For Authors
- About
- Become a Reviewer
Past Issue in 2024
Volume: 14, Issue: 13
Biochemistry
Analysis and Quantification of the Mitochondrial–ER Lipidome
Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress.Key features• Analysis and quantification of lipids in mitochondria–ER fraction through liquid chromatography–tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics).• LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems.• LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.
Biological Engineering
A Protocol for Preparing Mucoadhesive Emulsion Microgels and Assessing Their Mucoadhesion Properties In Vitro
Intravesical instillation is an efficient therapeutic technique based on targeted administration of a drug directly into the lesion for the treatment of bladder diseases. This is an alternative to traditional systemic administration of drugs. However, this technique requires repeated procedures, which can lead to even greater inflammation and infection of the urethra. To date, novel systems that allow prolonged drug retention in the bladder cavity are actively being developed. We recently reported a targeted drug delivery system based on the mucoadhesive emulsion microgels consisting of the natural component whey protein isolate. Such micron-sized carriers possess high loading capacity, a prolonged drug release profile, and efficient mucoadhesive properties to the bladder urothelium. As a continuation of this work, we present a protocol for the synthesis of mucoadhesive emulsion microgels. Detailed procedures for preparing precursor solutions as well as studying the physico-chemical parameters of microgels (including loading capacity and drug release rate) and the mucoadhesive properties using the model of porcine bladder urothelium are discussed. Precautionary measures and nuances that are worth paying attention to during each experimental stage are given as well.
Cell Biology
Quantification of Autophagosomes in Human Fibroblasts Using Cyto-ID® Staining and Cytation Imaging
As an essential process for the maintenance of cellular homeostasis and function, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles; therefore, dysregulation of autophagy in humans can lead to a variety of diseases. The link between impaired autophagy and disease highlights the need to investigate possible interventions to address dysregulations. One possible intervention is hyperthermia, which is described in this protocol. To investigate these interventions, a method for absolute quantification of autophagosomal compartments is required that allows comparison of autophagosomal activity under different conditions. Existing methods such as western blotting and immunohistochemistry for analysing the location and relative abundance of intracellular proteins associated with autophagy, or transmission electron microscopy (TEM), which are either very time-consuming, expensive, or both, are less suitable for this purpose. The method described in this protocol allows the absolute quantification of autophagosomes per cell in human fibroblasts using the CYTO-ID® Autophagy Detection Kit after heat therapy compared to a control. The Cyto-ID® assay is based on the use of a specific dye that selectively stains autophagic compartments, combined with an additional Hoechst 33342 dye for nuclear staining. The subsequent recognition of these stained compartments by the Cytation Imager enables the software to determine the number of autophagosomes per nucleus in living cells. Additionally, this absolute quantification uses an image-based method, and the protocol is easy to use and not time-consuming. Furthermore, the method is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance.
Microbiology
Imaging Assays to Detect DNA Damage in Trypanosome Parasites Using γH2A
Diseases caused by trypanosomatid parasites remain a significant unmet medical need for millions of people globally. Trypanosomatid parasites such as Trypanosoma cruzi and subspecies of Trypanosoma brucei cause Chagas disease and human African trypanosomiasis (HAT), respectively. Although efforts to find novel treatments have been successful for HAT, Chagas disease is still treated with decades-old therapies that suffer from long treatment durations and severe safety concerns. We recently described the identification and characterization of the cyanotriazole compound class that kills trypanosomes, in vitro and in vivo, by selective inhibition of the trypanosome nuclear topoisomerase II enzyme. To evaluate whether inhibition of the topoisomerase II enzyme led to parasite death due to lethal double-strand DNA breaks, we developed assays for detecting DNA damage in both intracellular amastigotes of T. cruzi and bloodstream-form T. brucei by using the canonical DNA damage marker γH2A. Herein, this article describes the protocols for detecting DNA damage using an immunofluorescence assessment of γH2A by microscopy in trypanosome parasites.
Neuroscience
Bilateral Common Carotid Artery Stenosis in Mice: A Model of Chronic Cerebral Hypoperfusion-Induced Vascular Cognitive Impairment
Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia. Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI. Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable. Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm. To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils. This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture. Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI.
Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry
Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia–derived progenitor cells that arise after treatment with PNU-282987.
Plant Science
Phylogenomics of Plant NLR Immune Receptors to Identify Functionally Conserved Sequence Motifs
In recent years, the increase in genome sequencing across diverse plant species has provided a significant advantage for phylogenomics studies, allowing the analysis of one of the most diverse gene families in plants: nucleotide-binding leucine-rich repeat receptors (NLRs). However, due to the sequence diversity of the NLR gene family, identifying key molecular features and functionally conserved sequence patterns is challenging through multiple sequence alignment. Here, we present a step-by-step protocol for a computational pipeline designed to identify evolutionarily conserved motifs in plant NLR proteins. In this protocol, we use a large-scale NLR dataset, including 1,862 NLR genes annotated from monocot and dicot species, to predict conserved sequence motifs, such as the MADA and EDVID motifs, within the coiled-coil (CC)-NLR subfamily. Our pipeline can be applied to identify molecular signatures that have remained conserved in the gene family over evolutionary time across plant species.
Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis
CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene.
Correction
