This chapter describes an imaging flow cytometry technique, a fusion of microscopy and flow cytometry principles, to precisely measure and quantify EBIs in samples harvested from mouse bone marrow. Adapting this method to other tissues, including the spleen, or to other species, is contingent upon the existence of fluorescent antibodies that are particular to both macrophages and erythroblasts.

Fluorescence methods provide a common approach to the investigation of marine and freshwater phytoplankton communities. Determining various microalgae populations based on autofluorescence signals poses a significant analytical challenge. Our novel approach to tackling this issue involved utilizing the versatility of spectral flow cytometry (SFC) and generating a matrix of virtual filters (VFs), allowing for a detailed examination of autofluorescence spectra. This matrix allowed a study of the varying spectral emission patterns of algae species, yielding the discrimination of five key algal taxonomic groups. Following the acquisition of these results, a subsequent application was the tracing of specific microalgae taxa within the diverse mixtures of laboratory and environmental algal populations. Integrated analysis of single algal events and unique spectral emission fingerprints, alongside light-scattering parameters, enables the classification of different microalgal groups. A novel protocol for evaluating the quantity of heterogeneous phytoplankton populations at the single-cell level is presented, including the monitoring of phytoplankton blooms with a virtual filtering technique performed on a spectral flow cytometer (SFC-VF).

Spectral flow cytometry, a new technology, allows for high-precision measurements of fluorescent spectra and light-scattering characteristics in diverse cell populations. Highly advanced instrumentation allows the concurrent determination of up to 40+ fluorescent dyes with overlapping emission spectra, the segregation of autofluorescent signals within the stained specimens, and the comprehensive investigation of diverse autofluorescence in various cell types, from mammalian cells to chlorophyll-containing organisms like cyanobacteria. The paper reviews the history of flow cytometry, contrasts conventional and spectral cytometers, and examines several applications enabled by spectral flow cytometry.

An epithelial barrier's innate immune system, in response to the invasion of pathogens such as Salmonella Typhimurium (S.Tm), initiates inflammasome-induced cell death. Ligands associated with pathogens or damage are recognized by pattern recognition receptors, subsequently leading to inflammasome activation. The cumulative effect is to constrain bacterial presence within the epithelium, to restrict damage to the barrier, and to prevent inflammatory tissue harm. The expulsion of dying intestinal epithelial cells (IECs) from the epithelial lining, characterized by the permeabilization of cell membranes at some stage, plays a crucial role in mediating pathogen restriction. Real-time study of inflammasome-dependent mechanisms is possible using intestinal epithelial organoids (enteroids), which enable high-resolution imaging in a stable focal plane when cultured as 2D monolayers. These protocols outline the procedures for establishing murine and human enteroid-derived monolayers, as well as for observing, via time-lapse imaging, IEC extrusion and membrane permeabilization subsequent to S.Tm-induced inflammasome activation. The protocols are adaptable to examining alternative pathogenic triggers, alongside genetic and pharmacological manipulations of the relevant pathways.

Infectious and inflammatory agents can trigger the activation of inflammasomes, which are multiprotein complexes. Inflammasome activation triggers the process of maturation and secretion of pro-inflammatory cytokines, and additionally, the characteristic form of lytic cell death, namely pyroptosis. Pyroptosis is typified by the complete release of cellular material into the extracellular space, thereby boosting the local innate immune reaction. The alarmin high mobility group box-1 (HMGB1) stands out as a particularly noteworthy component. Extracellular HMGB1, a robust instigator of inflammation, leverages multiple receptors to initiate and sustain the inflammatory cascade. This protocol series details the induction and evaluation of pyroptosis in primary macrophages, emphasizing HMGB1 release assessment.

Cell permeabilization, a hallmark of pyroptosis, an inflammatory form of cell death, is brought about by the cleavage and activation of gasdermin-D, a pore-forming protein, by the activated caspase-1 or caspase-11. The observable features of pyroptosis include cell swelling and the liberation of inflammatory cytosolic elements, once thought to be caused by colloid-osmotic lysis. Our earlier in vitro observations demonstrated that pyroptotic cells, to our surprise, do not lyse. Calpain's effect on vimentin, leading to a degradation of intermediate filaments, was shown to contribute to cell fragility and susceptibility to rupture under exterior pressure. medical comorbidities Nonetheless, if, per our observations, cells do not expand due to osmotic pressures, what, then, precipitates the disintegration of the cell? During pyroptosis, the loss of intermediate filaments is coupled with the disruption of other cytoskeletal components, including microtubules, actin, and the nuclear lamina; the mechanisms behind these losses and the functional consequences of these cytoskeletal alterations, however, remain unclear. TAK-861 cost To advance the understanding of these processes, we detail here the immunocytochemical techniques used to identify and quantify cytoskeletal damage during pyroptosis.

The inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11), activated by inflammasomes, trigger a chain reaction of cellular events resulting in proinflammatory cell death, also known as pyroptosis. Gasdermin D's proteolytic cleavage event results in the generation of transmembrane pores, which subsequently allow the release of mature interleukin-1 and interleukin-18 cytokines. Plasma membrane Gasdermin pores allow calcium to enter, initiating lysosomal fusion with the cell surface, releasing their contents into the extracellular environment through a process called lysosome exocytosis. This chapter describes procedures to measure calcium flux, lysosome release, and membrane disruption after the inflammatory caspases are activated.

Inflammation in autoinflammatory illnesses and the host's response to infection are substantially influenced by the interleukin-1 (IL-1) cytokine. Within cellular structures, IL-1 is stored in a dormant state, necessitating the proteolytic elimination of an amino-terminal fragment for its binding to the IL-1 receptor complex and subsequent pro-inflammatory activity. Inflammasome-activated caspase proteases are typically responsible for this cleavage event, although microbe and host proteases can produce distinct active forms. The post-translational regulation of IL-1, and the consequent multiplicity of resultant products, can create hurdles in the evaluation of IL-1 activation. The accurate and sensitive measurement of IL-1 activation in biological samples is the subject of this chapter, which details the methodologies and critical controls.

Within the Gasdermin family, Gasdermin B (GSDMB) and Gasdermin E (GSDME) are notable members, possessing a highly conserved Gasdermin-N domain. This domain is critically involved in the execution of pyroptotic cell death, a process characterized by plasma membrane perforation originating from within the cell's interior. Autoinhibition of GSDMB and GSDME prevails in the resting state, demanding proteolytic cleavage to liberate their pore-forming capabilities, which are otherwise masked by their C-terminal gasdermin-C domain. The activation of GSDMB hinges on the cleavage by granzyme A (GZMA) from cytotoxic T lymphocytes or natural killer cells, in contrast to GSDME's activation by caspase-3, which follows various apoptotic stimuli. The methods for inducing pyroptosis, specifically focusing on the cleavage of GSDMB and GSDME, are described in this work.

Gasdermin proteins, excluding DFNB59, are the agents responsible for pyroptotic cell demise. Lytic cell death results from an active protease's action on gasdermin. Gasdermin C (GSDMC) is a target for caspase-8 cleavage, in response to the macrophage's secretion of TNF-alpha. The process of cleavage liberates the GSDMC-N domain, which then oligomerizes and forms pores in the plasma membrane. Reliable markers for GSDMC-mediated cancer cell pyroptosis (CCP) include GSDMC cleavage, LDH release, and plasma membrane translocation of the GSDMC-N domain. We demonstrate the techniques used in the examination of CCP, mediated by GSDMC.

Gasdermin D's function is indispensable in orchestrating the pyroptosis response. Under resting conditions, the cytosol harbors an inactive gasdermin D. Following the activation of the inflammasome, gasdermin D is processed and oligomerized, forming membrane pores that trigger pyroptosis and release mature IL-1β and IL-18. mucosal immune To evaluate gasdermin D's function, biochemical approaches to analyzing the activation states of gasdermin D are indispensable. We detail the biochemical procedures for evaluating gasdermin D's processing, oligomerization, and inactivation through small molecule inhibitors.

The immunologically silent cell death process, apoptosis, is most commonly driven by caspase-8. However, emerging investigation suggested that pathogen-mediated inactivation of innate immune signaling, like that occurring during Yersinia infection in myeloid cells, promotes the interaction of caspase-8 with RIPK1 and FADD, triggering a pro-inflammatory, death-inducing complex. Caspase-8, in these conditions, effects cleavage of the pore-forming protein gasdermin D (GSDMD), resulting in a lytic form of cell death, recognized as pyroptosis. We present here a detailed protocol for inducing caspase-8-dependent GSDMD cleavage in murine bone marrow-derived macrophages (BMDMs) infected with Yersinia pseudotuberculosis. In particular, we outline the procedures for harvesting and culturing BMDMs, preparing Yersinia for inducing type 3 secretion systems, infecting macrophages, assessing lactate dehydrogenase release, and performing Western blot validations.