细胞生物学

Cell viability and cytotoxicity assays are commonly used to investigate protein function and to evaluate drug efficacy in cancer and other disease models. Cytotoxicity is the measure of dead or damaged cells and is often quantified using assays based on cellular characteristics such as membrane integrity or mitochondrial metabolism. However, these assays are typically limited to endpoint analysis and lack emulation of physiological conditions. The IncuCyte Live and Dead Cell assay described here leverages common cell permeability methodologies but uses fluorescence microscopy channels to image both live and dead cells over time and phase microscopy channels to measure confluency. Cytotox green reagent is a cell membrane–impermeable dye that can only be taken up by cells with poor cell membrane integrity. NucLight rapid red dye is a cell membrane–permeable nuclear dye that can be taken up by all cells. Based on dye uptake and fluorescence intensity, the IncuCyte software can be used to analyze images for live and dead cell detection and quantification. Phase microscopy is used to determine confluency and can be further quantified using the IncuCyte software. We provide an application of this assay, using it to calculate IC₅₀ and EC₅₀ values for the assessment of drug efficacy.

Neurons are highly polarized cells, with axons that may innervate distant target regions. In the brain, basal forebrain cholinergic neurons (BFCNs) possess extensive axons that project to several target regions such as the cortex, hippocampus, and amygdala, and may be exposed to a specific microenvironment in their axon targets that may have retrograde effects on neuronal health. Interestingly, BFCNs express the pan-neurotrophin receptor p75NTR throughout life while also concomitantly co-expressing all Trk receptors, making them capable of responding to both mature and precursor neurotrophins to promote survival or apoptosis, respectively. Levels of these trophic factors may be modulated in the BFCN axon or soma microenvironment under neurodegenerative conditions such as seizure and brain injury. In this protocol, BFCNs are established in microfluidic devices for compartmental culture, with the aim of studying the effects of axon- or soma-specific stimulation of BFCNs for an in vitro representation of distal axon vs. soma environments as seen in vivo. This study further establishes a novel method of tracing and imaging live BFCNs exposed to stimuli in their distal axons with the aim of assessing retrograde cell death. The in vitro compartmental culture system of BFCNs that allows live imaging may be applied to investigate various effects of axon- or soma-specific stimuli that affect BFCN health, maintenance, and death, to model events that occur in the context of brain injury and neurodegenerative disorders.

In modern science, the exchange of scientific material between different institutions and collaborating working groups constitutes an indispensable endeavor. For this purpose, bacterial strains are frequently shipped to collaborators to advance joint research projects. Bacterial strains are usually safely shipped as cultures on solid medium, whereas the shipment of liquid cultures requires specific safety measures due to the risk of leakage. Cyanobacterial cultures are frequently maintained as liquid stock cultures, and this problem typically arises. This protocol describes a new method for the shipment of liquid cyanobacterial stock cultures by agarose gel embedding (SCAGE). More specifically, a cyanobacterial culture is mixed with low-melting agarose and cast into sterile plastic bags, resulting in a thin, solid cyanobacterial agarose gel (cyanogel) that can be easily shipped. After delivery, subsequent regeneration of the cyanogel material in liquid media results in full recovery of the examined bacterial strains. Thus, the packaging method devised in the present study comprises an innovative technique to facilitate the shipment of bacterial strains, whilst eliminating previously encountered issues like cell culture leakage.

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.

The isolation of intact single adult cardiomyocytes from model animals, mouse and rat, is an essential tool for cardiac molecular and cellular research. While several methods are reported for adult mouse cardiomyocyte isolation, the viability and yield of the isolated cells have been variable. Here, we describe step-by-step protocols for high viability and yield cardiomyocyte isolation from mouse and rat, based on the use of a stable pressure Langendorff perfusion system. After the animal is euthanized or terminally anesthetized, the heart is removed from the chest and subject to Langendorff perfusion. Then, the heart is digested by perfusion with collagenase and hyaluronidase. After thorough digestion, the cardiomyocytes are dispersed and gradually recovered, the extracellular Ca²⁺ concentration adjusted, and cells are then ready for use. This protocol will facilitate research that requires isolated adult mouse and rat cardiomyocytes.

When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response.

Graphic abstract:

In-vitro simulation of restimulation-induced cell death in activated human T cells.

Apoptotic cell death eliminates unhealthy cells and maintains homeostatic cell numbers within tissues. Epithelia, which serve as fundamental tissue barriers for the body, depend on a physical expulsion of dying cells (apoptotic cell extrusion) to remain sealed and intact. Apoptotic cell extrusion has been widely studied over recent years, with researchers using various approaches to induce apoptotic cell death. Unfortunately, the majority of chemical-based approaches for cell death induction rely on sporadically occurring apoptosis and extrusion, making imagining lengthy, often unsuccessful, and difficult to capture in high-quality images because of the frequent frame sampling needed to visualise the key molecular processes that drive extrusion. Here, we present a protocol that describes steps needed for laser-mediated induction of apoptosis in a cell of choice, followed by imaging of apoptotic extrusion in confluent monolayers of epithelial cells. Moreover, we provide the description of a new approach involving the mixing of labelled and unlabelled cells. In particular, this protocol characterises how cells surrounding apoptotic cells behave, with high spatial and temporal resolution. This can be achieved without the optical interference that apoptotic cells cause as they are physically expelled from the monolayer and move out of focus for imaging. Finally, the protocol is accompanied by detailed procedures describing cell preparation for apoptotic extrusion experiments, as well as post-acquisition analysis required to evaluate rates of successful extrusion.

The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca²⁺ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca²⁺ entry through the plasma membrane leading to Ca²⁺-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca²⁺ flux, and cell viability elicited by HK2pep treatment of tumor cells.

Graphical abstract:

Flowchart for the analysis of the effects of HK2 detachment from MAMs.

The in vivo toxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) or their hydrogels such as with hydroxyethyl-methacrylate (HEMA) (pHEMA@SBAMs or pHEMA@CoMeDs) are evaluated by the brine shrimp assay. Thus individuals of Artemia salina larvae are incubated in saline solutions with SBAMs, CoMeDs, pHEMA@SBAMs or pHEMA@CoMeDs or without for 24 h. The toxicity is then determined in terms of the mortality rate of brine shrimp larvae. Brine shrimp assay is a low cost, safe, no required feeding during the assay, while it requiring only a small amount of the tested agent.

This protocol details the construction of a simple, low-cost, small-scale, multiplex chemostat system designed for the continuous cultivation of microorganisms in suspension (i.e., bacteria, yeast, microalgae). The continuous culture device can operate at a working volume of 25 ml and allows the run of 8 chemostats in parallel by a single person. It provides a platform for parallel, long-term studies of evolution and adaptation of microorganisms under the stress of antimicrobial agents and/or toxic pollutants. The system complies with the varied needs of researchers for an accessible, highly-throughput and reliable tool that is nevertheless easy to construct, use and operate, and not demanding of space, materials, medium supply and workload. Here, we also validate the use of this system to generate de novo resistance towards a novel antimicrobial and a commonly used antibiotic in an antimicrobial-sensitive model organism. We believe that this "Do It Yourself" (DIY) system may constitute a useful tool to address the global problem of antibiotic resistance and to develop non-antibiotic based therapies.