Editor
Chiara Ambrogio
  • Faculty, Department of Molecular Biotechnology and Health Sciences, University of Torino
Research fields
  • Cancer Biology, Cell Biology, Molecular Biology
Total Internal Reflection Fluorescence (TIRF) Single-Molecule Assay to Analyze the Motility of Kinesin
Authors:  Tomoki Kita and Shinsuke Niwa, date: 12/20/2024, view: 413, Q&A: 0

The motile parameters of kinesin superfamily proteins are fundamental to intracellular transport. Single-molecule motility assays using total internal reflection fluorescence (TIRF) microscopy are a gold standard technique for measuring the motile parameters of kinesin motors. With this technique, one can evaluate the velocity, run length, and binding frequency of kinesins on microtubules by directly observing their motility. This protocol provides a comprehensive procedure for single molecule assays of kinesins, including the preparation of labeled microtubules, the measurement of kinesin motility via TIRF microscopy, and the quantification of kinesin motor parameters.

Isolation and Enrichment of Major Primary Neuroglial Cells from Neonatal Mouse Brain
Authors:  Santosh Kumar Samal, Madhav Sharma and Jayasri Das Sarma, date: 01/20/2024, view: 1407, Q&A: 0

The central nervous system (CNS) relies on the complex interaction of neuroglial cells to carry out vital physiological functions. To comprehensively understand the structural and functional interplay between these neuroglial cells, it is essential to establish an appropriate in vitro system that can be utilized for thorough investigation. Traditional protocols for establishing primary neuronal and mixed glial cultures from prenatal mice or neural stem cells require sacrificing pregnant mice and have the drawback of yielding only specific types of cells. Our current protocol overcomes these drawbacks by utilizing the brain from day-0 pups to isolate CNS resident neuroglial cells including astrocytes, microglia, oligodendrocytes [oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes], and meningeal fibroblasts, as well as hippocampal neurons, avoiding sacrificing pregnant mice, which makes this procedure efficient and cost effective. Furthermore, through this protocol, we aim to provide step-by-step instructions for isolating and establishing different primary neuroglial cells and their characterization using cell-specific markers. This study presents an opportunity to isolate, culture, and establish all major CNS resident cells individually. These cells can be utilized in various cell-based and biochemical assays to comprehensively investigate the cell-specific roles and behaviors of brain resident cells in a reductionist approach.


Key features

• Efficient isolation of major neuroglial cells like meningeal fibroblasts, neurons, astrocytes, oligodendrocytes, and microglia from a single day-0 neonatal mouse pup’s brain.

• Circumvents the sacrifice of pregnant female mice.

• Acts as a bridging experimental method between secondary cell lines and in vivo systems.

• Isolated cells can be used for performing various cell-based and biochemical assays.


Graphical overview



Steps for isolation of meningeal fibroblast and neuroglial cells from day 0 pups of mice (Created using BioRender.com)


Automated 384-well SYBR Green Expression Array for Optimization of Human Induced Pluripotent Stem Cell Differentiation
Authors:  Max Y. Chen, Laurin Heinrich, Faria Zafar, Kamilla Sedov and Birgitt Schuele, date: 06/05/2023, view: 884, Q&A: 0

Cell populations and tissues exhibit unique gene expression profiles, which allow for characterizing and distinguishing cellular subtypes. Monitoring gene expression of cell type–specific markers can indicate cell status such as proliferation, stress, quiescence, or maturation. Quantitative reverse transcriptase PCR (qRT-PCR) allows quantifying RNA expression of cell type–specific markers and distinguishing one cell type from another. However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive. Processing RNA sequencing data can take several weeks, which is not optimal for quality control and monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type.

A more cost-effective assay is based on SYBR Green technology. SYBR Green is a nucleic acid dye that binds to double-stranded DNA, absorbs blue light at 497 nm, and emits green light at 520 nm up to 1,000-fold upon intercalation with double-stranded DNA. Amplification of a region of interest can be quantified based on the level of fluorescence intensity when normalized to a housekeeping gene and compared to control conditions. Previously, we established a SYBR Green qRT-PCR protocol to characterize samples using a limited set of markers plated on a 96-well plate.

Here, we optimize the process and increase throughput to a 384-well format and compare mRNA expression to distinguish iPSC-derived neuronal subtypes from each other by increasing the number of genes, cell types, and differentiation time points. In this protocol, we develop the following: i) using the command-line version of Primer3 software, we design primers more easily and quickly for the gene of interest; ii) using a 384-well plate format, electronic multichannel pipettes, and pipetting robots, we analyze four times more genes on a single plate while using the same volume of reagents as in a 96-well plate. The advantages of this protocol are the increased throughput of this SYBR Green assay while limiting pipetting errors/inconsistencies, reagent use, cost, and time.


Graphical overview



Figure 1. Overall optimized SYBR Green qRT-PCR workflow. (A) Primers are designed through the command-line version of Primer3. The program takes a couple of files as arguments: 1) an input file containing a sequence of the region of interest and a target, and 2) settings file with custom settings and primer picking conditions. The results are saved to a text file, checked for secondary and tertiary structures, then synthesized. (B) Primers are then plated using either multichannel pipettes with a pipetting aid or an automated pipetting robot. Plates are left to dry at room temperature and can be stored for an indefinite time. (C) Meanwhile, RNA is extracted from cell samples, reverse-transcribed into cDNA, then plated onto pre-coated 384-well plates. SYBR Green qRT-PCR is run and analyzed with QuantStudio software and Microsoft Excel.
Quantifying Single and Dual Channel Live Imaging Data: Kymograph Analysis of Organelle Motility in Neurons
Authors:  Laura Digilio, Lloyd P. McMahon, Alois Duston, Chan Choo Yap and Bettina Winckler, date: 05/20/2023, view: 995, Q&A: 0

Live imaging is commonly used to study dynamic processes in cells. Many labs carrying out live imaging in neurons use kymographs as a tool. Kymographs display time-dependent microscope data (time-lapsed images) in two-dimensional representations showing position vs. time. Extraction of quantitative data from kymographs, often done manually, is time-consuming and not standardized across labs. We describe here our recent methodology for quantitatively analyzing single color kymographs. We discuss the challenges and solutions of reliably extracting quantifiable data from single-channel kymographs. When acquiring in two fluorescent channels, the challenge becomes analyzing two objects that may co-traffic together. One must carefully examine the kymographs from both channels and decide which tracks are the same or try to identify the coincident tracks from an overlay of the two channels. This process is laborious and time consuming. The difficulty in finding an available tool for such analysis has led us to create a program to do so, called KymoMerge. KymoMerge semi-automates the process of identifying co-located tracks in multi-channel kymographs and produces a co-localized output kymograph that can be analyzed further. We describe our analysis, caveats, and challenges of two-color imaging using KymoMerge.

In vivo Drug Screening to Identify Anti-metastatic Drugs in Twist1a-ERT2 Transgenic Zebrafish
Authors:  Joji Nakayama, Hideki Makinoshima and Zhiyuan Gong, date: 05/20/2023, view: 547, Q&A: 1

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.


Graphical overview




Timing

Day 0: Zebrafish spawning

Day 8: Primary tumor induction

Day 11: Chemical treatment

Day 11.5: Metastatic dissemination induction in the presence of a test chemical

Day 16: Data analysis

Fluorescence Assays for Real-Time Tracking of Cell Surface Protein Internalization and Endosomal Sorting in Axons of Primary Mouse Hippocampal Neurons
Authors:  Tai Chaiamarit, Yin Wu, Adriaan Verhelle and Sandra E. Encalada, date: 04/05/2023, view: 862, Q&A: 0

The trafficking and sorting of proteins through the secretory-endolysosomal system is critical for the proper functioning of neurons. Defects in steps of these pathways are associated with neuronal toxicity in various neurodegenerative disorders. The prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored protein that follows the secretory pathway before reaching the cell surface. Following endocytosis from the cell surface, PrP sorts into endosomes and lysosomes for further recycling and degradation, respectively. A few detailed protocols using drug treatments and fluorescent dyes have previously allowed the tracking of PrP trafficking routes in real time in non-neuronal cells. Here, we present a protocol optimized for primary neurons that aims to monitor and/or manipulate the trafficking and sorting of PrP particles at several steps during their secretory-endolysosomal itineraries, including (a) ER export, (b) endocytosis, (c) lysosomal degradation, and (d) accumulation in axonal endolysosomes. These primary neuron live assays allow for the robust quantitation of accumulation and/or degradation of PrP or of other membrane-associated proteins that transition from the ER to the Golgi via the cell surface.


Graphical abstract


Isolation of Immunocomplexes from Zebrafish Brain
Authors:  Jennifer Carlisle Michel and Adam C. Miller, date: 04/05/2023, view: 429, Q&A: 0

Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.

Gastrulation Screening to Identify Anti-metastasis Drugs in Zebrafish Embryos
Authors:  Joji Nakayama, Hideki Makinoshima and Zhiyuan Gong, date: 10/05/2022, view: 1039, Q&A: 0

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.


Graphical abstract:




CRISPR/Cas9-mediated LRP10 Knockout in HuTu-80 and HEK 293T Cell Lines
Authors:  Martyna M. Grochowska, Vincenzo Bonifati and Wim Mandemakers, date: 10/05/2022, view: 2226, Q&A: 0

Loss-of-function (LoF) variants in the low-density lipoprotein receptor–related protein 10 gene (LRP10) have been recently implicated in the development of neurodegenerative diseases, including Parkinson's disease (PD), PD dementia (PDD), and dementia with Lewy bodies (DLB). However, despite the genetic evidence, little is known about the LRP10 protein function in health and disease. Here, we describe a detailed protocol to efficiently generate a LRP10 LoF model in two independent LRP10-expressing cell lines, HuTu-80 and HEK 293T, using the CRISPR/Cas9 genome-editing tool. Our method efficiently generates bi-allelic LRP10 knockout (KO), which can be further utilized to elucidate the physiological LRP10 protein function and to model some aspects of neurodegenerative disorders.


Graphical abstract:



CRISPR/Cas9 workflow for the generation of the LRP10 KO. (1) Designed single guide RNA (sgRNA) is cloned into CRISPR/Cas9 px458 plasmid. (2) Cells are transfected with the CRISPR/Cas9 plasmid containing sgRNA. (3) Two days post transfection, cells are dissociated and sorted as single cells by fluorescence-activated cell sorting (FACS). (4) After several weeks, expanded clonal lines are (5) verified with Sanger sequencing for the presence of INDELs (insertions or deletions), RT-qPCR for the amounts of LRP10 mRNA transcript, and Western blotting for the analysis of the LRP10 protein levels.


Identification of Socially-activated Neurons
Authors:  Mary L. Phillips and Lucas Pozzo-Miller, date: 09/05/2020, view: 4983, Q&A: 0
Determining the neuronal circuitry responsible for specific behaviors is a major focus in the field of neurobiology. Activity-dependent immediate early genes (IEGs), transcribed and translated shortly after neurons discharge action potentials, have been used extensively to either identify or gain genetic access to neurons and brain regions involved in such behaviors. By using immunohistochemistry for the protein product of the IEG c-Fos combined with retrograde labeling of specific neuronal populations, precise experimental timing, and identical data acquisition and processing, we present a method to quantitatively identify specific neuronal subpopulations that were active during social encounters. We have previously used this method to show a stronger recruitment of ventral hippocampal neurons that project to the medial prefrontal cortex, compared to those that project to the lateral hypothalamus, following social interactions. After optimization of surgeries for the injection of retrograde tracers, this method will be useful for the identification and mapping of neuronal populations engaged in many different behaviors.
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