Importantly, the status of cellular membranes, particularly at the single-cell level, concerning their state or order, are often of considerable interest. In this initial description, we explain the use of Laurdan, a membrane polarity-sensitive dye, to optically measure the arrangement order of cellular groups over a wide temperature interval from -40°C to +95°C. This method provides a way to ascertain the position and width of biological membrane order-disorder transitions. Then, we demonstrate that the membrane order distribution across a group of cells empowers correlation analysis of membrane order and permeability. This third procedure, combining this method with standard atomic force spectroscopy, enables the quantitative determination of a connection between the overall effective Young's modulus of living cells and the order within their membranes.

Intracellular pH (pHi) is indispensable to regulating a broad spectrum of biological functions, each of which operates optimally at specific pH ranges inside the cell. Minute pH adjustments can influence the modulation of various molecular processes, including enzymatic activities, ion channel operations, and transporter functions, all of which are essential to cellular processes. Various optical methods utilizing fluorescent pH indicators remain integral parts of the continuously evolving techniques used for quantifying pHi. A method for quantifying the cytosolic pH of Plasmodium falciparum blood-stage parasites is presented here, utilizing the pH-sensitive fluorescent protein pHluorin2, which is introduced into the parasite's genome, and analyzed using flow cytometry.

Variables such as cellular health, functionality, response to environmental stimuli, and others impacting cell, tissue, or organ viability are clearly discernible in the cellular proteomes and metabolomes. Omic profiles are constantly adapting, even within typical cellular processes, ensuring cellular balance. This adaptation is driven by small environmental adjustments and the need to maintain optimal cell viability. Factors like cellular aging, disease response, and environmental adaptation, as well as other influential variables, are identifiable using proteomic fingerprints, ultimately informing our understanding of cellular viability. To ascertain proteomic changes, both qualitatively and quantitatively, a range of proteomic approaches are available. Isobaric tags for relative and absolute quantification (iTRAQ), a frequently employed technique, will be the focus of this chapter for examining shifts in proteomic expression within cells and tissues.

The contractile power of muscle cells, crucial for movement, is truly remarkable. Skeletal muscle fibers maintain full viability and functionality when their excitation-contraction (EC) coupling mechanisms are completely operational. For proper action potential generation and conduction, intact membrane integrity, complete with polarized membranes and functional ion channels, is essential. At the fiber's triad's level, the electrochemical interface is critical for triggering sarcoplasmic reticulum calcium release, which subsequently activates the contractile apparatus's chemico-mechanical interface. The final and visible result of a short electrical pulse stimulation is a twitching contraction. Intact and viable myofibers are critical for many biomedical studies that focus on single muscle cells. Consequently, a basic global screening method, consisting of a short electrical pulse applied to individual muscle fibers, and evaluating the visible contraction, would hold substantial value. A detailed, step-by-step approach, outlined in this chapter, describes the isolation of complete single muscle fibers from fresh muscle tissue through an enzymatic digestion process, complemented by a method for assessing twitch response and viability. A do-it-yourself stimulation pen, offering unique capabilities for rapid prototyping, comes with a fabrication guide to avoid the expenses of specialized commercial equipment.

The capacity of numerous cell types to thrive hinges critically on their adaptability to mechanical environments and fluctuations. Research into cellular mechanisms for detecting and responding to mechanical forces and the pathophysiological divergences in these systems has seen a notable rise in recent years. In numerous cellular processes, including mechanotransduction, the important signaling molecule calcium (Ca2+) plays a critical role. Experimental techniques for investigating live cellular calcium signaling under mechanical strain reveal previously unobserved mechanisms of cell mechanical response. In-plane isotopic stretching of cultured cells on elastic membranes allows for live assessment of intracellular Ca2+ levels using fluorescent calcium indicator dyes, all on a single-cell basis. Samuraciclib ic50 A procedure for functionally screening mechanosensitive ion channels and related drug tests is shown using BJ cells, a foreskin fibroblast cell line which readily responds to acute mechanical inputs.

Neural activity, spontaneous or evoked, can be measured using microelectrode array (MEA) technology, a neurophysiological method, to ascertain the attendant chemical impacts. To evaluate cell viability in the same well, a multiplexed approach is used following the assessment of compound effects on multiple network function endpoints. Recent technological advancements permit the measurement of the electrical impedance of cells adhered to electrodes, greater impedance denoting a larger cell population. In longer exposure assays, the neural network's development supports rapid and frequent assessments of cell health, without compromising cell viability. Consistently, the LDH assay for cytotoxicity and the CTB assay for cell viability are applied only after the period of chemical exposure is completed because cell lysis is a requirement for these assays. Procedures for multiplexed screening of acute and network formations are presented in this chapter.

Through the method of cell monolayer rheology, a single experimental run yields quantification of average rheological properties for millions of cells assembled in a single layer. We detail a step-by-step approach for utilizing a modified commercial rotational rheometer to execute rheological measurements, determining the average viscoelastic properties of cells, while simultaneously ensuring the required level of precision.

Protocol optimization and validation, a prerequisite for fluorescent cell barcoding (FCB), are crucial for minimizing technical variations in high-throughput multiplexed flow cytometric analyses. In the field of measurement, FCB is extensively used for evaluating the phosphorylation state of certain proteins, and it also serves a valuable function in assessing cellular viability. Samuraciclib ic50 We introduce in this chapter the procedure for performing FCB combined with viability assessments on lymphocyte and monocyte populations, utilizing both manual and automated analytical techniques. Along with our work, we offer recommendations for refining and validating the FCB protocol for the analysis of clinical specimens.

Single-cell impedance measurements, which are noninvasive and label-free, allow for the characterization of the electrical properties of individual cells. In the current state of development, electrical impedance flow cytometry (IFC) and electrical impedance spectroscopy (EIS), while frequently utilized for impedance measurement, are typically applied individually to most microfluidic chips. Samuraciclib ic50 For high-efficiency single-cell electrical property measurement, we detail a method employing a single chip integrating both IFC and EIS techniques: single-cell electrical impedance spectroscopy. We posit that the integration of IFC and EIS strategies offers a unique methodology for optimizing the effectiveness of electrical property measurements of individual cells.

Cell biology research has benefited significantly from flow cytometry's long-standing role as a key instrument, enabling the detection and quantitative measurement of both physical and chemical characteristics of individual cells within a larger population. Thanks to recent advances in flow cytometry, nanoparticle detection is now possible. Mitochondria, as intracellular organelles, possess distinct subpopulations. Evaluation of these subpopulations is possible through examining the variations in their functional, physical, and chemical attributes, a process analogous to assessing different cell types. In assessing intact, functional organelles and fixed samples, the characteristics of size, mitochondrial membrane potential (m), chemical properties, and outer mitochondrial membrane protein expression are essential. Employing this method, multiparametric analysis of mitochondrial subpopulations is possible, in addition to the isolation of individual organelles for further analysis down to the single-organelle level. A fluorescence-activated mitochondrial sorting (FAMS) protocol is detailed, enabling the analysis and separation of mitochondria. This protocol employs fluorescent labeling and antibodies to isolate distinct mitochondrial subpopulations.

The preservation of neuronal networks depends crucially on the viability of neurons. Noxious modifications, already present in slight forms, such as the selective interruption of interneurons' function, which boosts excitatory activity inside a network, may already undermine the overall network's functionality. To evaluate neuronal network integrity, we implemented a network reconstruction strategy, inferring effective neuronal connectivity from live-cell fluorescence microscopy data of cultured neurons. Fluo8-AM, a fast calcium sensor, reports neuronal spiking with a high sampling rate (2733 Hz), allowing for the detection of rapid intracellular calcium increases, like those triggered by action potentials. Following a surge in recorded data, a machine learning-based algorithm set reconstructs the neuronal network. Via various parameters, including modularity, centrality, and characteristic path length, the topology of the neuronal network can thereafter be scrutinized. In essence, these parameters portray the network's structure and responsiveness to experimental manipulations, such as hypoxia, nutrient deprivation, co-culture setups, or the introduction of drugs and other interventions.