Successful Experiments Research Articles

Deep learning-based intraoperative guidance system for anatomical identification in laparoscopic surgery: A review

The power of Artificial Intelligence (AI) combined with the surgeons’ expertise leads to breakthroughs in surgical care, bringing new hope to patients. Utilizing deep learning-based computer vision techniques in surgical procedures will enhance the healthcare industry. Laparoscopic surgery holds excellent potential for computer vision due to the abundance of real-time laparoscopic recordings captured by digital cameras containing significant unexplored information. Furthermore, with computing power resources becoming increasingly accessible and Machine Learning methods expanding across various industries, the potential for AI in healthcare is vast. There are several objectives of AI’s contribution to laparoscopic surgery; one is an image guidance system to identify anatomical structures in real-time. However, few studies are concerned with intraoperative anatomy recognition in laparoscopic surgery. This study provides a comprehensive review of the current state-of-the-art semantic segmentation techniques, which can guide surgeons during laparoscopic procedures by identifying specific anatomical structures for dissection or avoiding hazardous areas. This review aims to enhance research in AI for surgery to guide innovations towards more successful experiments that can be applied in real-world clinical settings. This AI contribution could revolutionize the field of laparoscopic surgery and improve patient outcomes.

Kinetic Modelling of Ralstonia eutropha H16 Growth on Different Substrates

Due to environmental pollution and the depletion of fossil fuels, there is growing interest in the development and use of biofuels as environmentally friendly alternatives. One of the most promising biofuels is biohydrogen, hydrogen produced through sustainable processes using microorganisms such as bacteria and algae. One of the most interesting bacteria for hydrogen production is Ralstonia eutropha H16, known for its ability to produce oxygen-tolerant hydrogenases. These enzymes play a crucial role in biohydrogen metabolism and production. The aim of this work was to determine the optimal conditions (reactor type and synthetic medium composition) for the cultivation of R. eutropha H16. The culture media contained different concentrations of fructose and glycerol (mono- or double-substrate cultivation) and the experiments were carried out in a batch reactor. The initial experiments were carried out with 4 g/L fructose or glycerol in the culture medium at pH 7, T = 30 °C, and 120 rpm. The mathematical model, consisting of the growth kinetics (described by the Monod’s model) and the corresponding mass balances, was proposed. The developed model was validated using two independent experiments with different initial substrate concentrations: 2 g/L glycerol and fructose in one medium and 4 g/L fructose and 1 g/L glycerol in the second. In order to propose the optimal cultivation procedure for future research, the mathematical model simulations were performed for different reactor types (batch, fed-batch, and continuous stirred tank reactors) and different initial substrate concentrations. The most successful experiment was the one with 4 g/L glycerol, where γX = 0.485 ± 0.001 g/L of biomass was achieved. Further calculations showed that the most biomass would be produced at higher glycerol concentrations (at γG = 6.358 g/L, γX = 1.311 g/L should be achieved after 200 h of cultivation) and when using a fed-batch reactor (γX = 0.944 g/L after 200 h of cultivation).

Industrial hemp (Cannabis sativa L.) adapts to cadmium stress by reshaping rhizosphere fungal community

Increasing evidence indicates that plants under environmental stress can actively seek the help of microbes (‘cry-for-help’ hypothesis). However, empirical evidence underlying this strategy is limited under metal-stress conditions. Here, we employed integrated microbial community profiling in cadmium (Cd) polluted soil and culture-based methods to investigate the three-way interactions between the industrial hemp (Cannabis Sativa L.), rhizospheric microbes, and Cd stress. Results from the pot and three cycles of the successful hemp planting experiments showed that Cd stress significantly affected the composition of rhizosphere fungi in industrial hemp and induced enrichment of the fungal operational taxonomic unit (OTU)3 (Cladosporium). A representative of OTU3 (strain DM-2) was successfully isolated. In a hydroponic experiment, inoculation of DM-2 significantly increased the shoot length (by 25.84 %) and fresh weight (by 92.66 %) of hemp seedlings when compared to the absence of DM-2 under the Cd stress. The findings indicate that DM-2 inoculation could effectively alleviate the Cd stress in hemp seedlings. Metabolomic analysis of spent media with or without DM-2 revealed the association of DM-2 with the transformation of root exudates to melatonin, which may be a key chemical in plant-microbe interactions against abiotic stresses. The findings will inform efforts to manipulate the root microbiome to enhance plant growth in polluted environments.

Control of molecular weight distribution through photoresponsive RAFT polymerization with a temporal program-controlled system

The polymer dispersity index (PDI) is a vital metric for characterizing molecular weight distribution, significantly influencing polymer material performance. However, achieving precise control over PDI in polymer synthesis remains a substantial challenge. In this study, we introduce an approach for modifying the PDI of poly(methyl acrylate) (PMA) in switchable reversible addition-fragmentation chain transfer radical polymerizations (RAFT). The devised strategy involves the utilization of photoresponsive hexaarylbiimidazole (HABI) as a mediator, coupled with temporal programming, to cyclically deactivate and reactivate propagating radicals at distinct stages throughout the polymerization process. The precise timing of the light stimulus is facilitated through computer-controlled single-chip microcomputer technology, ensuring automatic modulation of the optical state and mitigating operational inaccuracies. By manipulating external light conditions, the PDI of PMAs can be systematically adjusted within the range of 1.80–2.59. Validation through successful chain-extension experiments and analysis via MALDI-TOF MS confirm the preservation of good chain-end fidelity across PMAs with varying PDIs fabricated through this methodology.

Eastern larch beetle (Dendroctonus simplex LeConte) as a potential threat to western (Larix occidentalis Nutt.) and subalpine larches (Larix lyalli Parl.)

Eastern larch beetle Dendroctonus simplex LeConte has been outbreaking for the past two decades in the Great Lakes region of North America, impacting approximately 400,000 hectares of eastern larch Larix laricina (tamarack) forest in Minnesota, United States, alone. Range expansions of some Dendroctonus species driven by climate change have occurred across North America as rising temperatures increase climatically suitable habitats across latitudes and elevations. The range of eastern larch beetle is currently sympatric to that of its host, eastern larch, but the insect could, in theory, eventually access ranges of other species of Larix native to North America. We compared host suitability of two potential novel hosts, western larch (Larix occidentalis Nutt.) and subalpine larch (Larix lyalli Parl.) against the beetle’s native host, eastern larch, using a reproductive success experiment conducted in cut bolts of all three species. We measured fertility, maternal gallery length, and offspring size and lipid content as metrics of reproductive success. We found beetles could reproduce in both novel Larix species, with subalpine larch having high levels of success compared to the beetle’s native host, eastern larch. These findings suggest that subalpine larch may be at risk should a range expansion of eastern larch beetle occur.

Review and Practical Guide for Getting Started With Single-Cell Proteomics.

Single-cell proteomics (SCP) has advanced significantly in recent years, with new tools specifically designed for the preparation and analysis of single cells now commercially available to researchers. The field is sufficiently mature to be broadly accessible to any lab capable of isolating single cells and performing bulk-scale proteomic analyses. In this review, we highlight recent work in the SCP field that has significantly lowered the barrier to entry, thus providing a practical guide for those who are newly entering the SCP field. We outline the fundamental principles and report multiple paths to accomplish the key steps of a successful SCP experiment including sample preparation, separation, and mass spectrometry data acquisition and analysis. We recommend that researchers start with a label-free SCP workflow, as achieving high-quality and quantitatively accurate results is more straightforward than label-based multiplexed strategies. By leveraging these accessible means, researchers can confidently perform SCP experiments and make meaningful discoveries at the single-cell level.

Mouse Testing Methods in Psychoneuroimmunology: Measuring Behavioral Responses.

The field of psychoneuroimmunology (PNI) aims to uncover the processes and consequences of nervous, immune, and endocrine system relationships. Behavior is a consequence of such interactions and manifests from a complex interweave of factors including immune-to-neural and neural-to-immune communication. Often the signaling molecules involved during a particular episode of neuroimmune activation are not known, but behavioral response provides evidence that bioactives such as neurotransmitters and cytokines are perturbed. Immunobehavioral phenotyping is a first-line approach when examining the neuroimmune system and its reaction to immune stimulation or suppression. Behavioral response is significantly more sensitive than direct measurement of a single specific bioactive and can quickly and efficiently rule in or out relevance of a particular immune challenge or therapeutic to neuroimmunity. Classically, immunobehavioral research was focused on sickness symptoms related to bacterial infection, but neuroimmune activation is now a recognized complication of diseases and disorders ranging from cancer to diabesity to Alzheimer's. Immunobehaviors include lethargy, loss of appetite, and disinterest in social activity/surrounding environment. In addition, neuroimmune activation can diminish physical activity, precipitate feelings of depression and anxiety, and impair cognitive and executive function. Provided is a detailed overview of behavioral tests frequently used to examine neuroimmune activation in mice with a special emphasis on pre-experimental conditions that can confound or prevent successful immunobehavioral experimentation.

Spent yeast reuse as inoculum: a strategy to boost cellulosic ethanol productivity

AbstractFor the implementation of an integrated biorefinery into a pulping mill, the valorization of bark through cellulosic ethanol production and simultaneously the valorization of the residual yeast from each fermentation run should be considered to increase sustainability and pulp and paper profitability. Spent yeast recycling during cellulosic ethanol production is an opportunity to reduce waste according to environmental issues for disposal at the industrial level, boosting the circular economy. This work evaluated the reuse of spent yeast in successive fermentation experiments at Erlenmeyer and bioreactor scales for cellulosic ethanol from Eucalyptus globulus bark. The spent yeast recovered through centrifugation at the end of each experiment was used as inoculum for subsequent batch fermentation. Across five sequential fermentations, wherein only a fraction of spent yeast was recycled, a discernible decline in ethanol volumetric productivity was observed throughout successive fermentation cycles. Conversely, employing the complete spent yeast for recycling along 11 consecutive trials resulted in a notable enhancement of ethanol productivity, as evidenced by a reduction in fermentation duration to approximately one-third remaining the conversion efficiency above 70%. Four successive experimental trials were conducted at the bioreactor scale, resulting in volumetric productivities ranging from 2.00 to 5.83 g L−1 h−1. The reuse of spent yeast in consecutive fermentation cycles demonstrably augmented productivity, thereby validating a promising strategy for the industrial-scale production of cellulosic ethanol. This significantly contributes to advances in the circular economy principles within the overarching framework of biorefinery operations. Graphical Abstract

Pilot-scale process development for recombinant adeno-associated virus (rAAV) production based on high-density Sf9 cell culture

BackgroundIn recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production.MethodsIn this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level.ConclusionsThis successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.

Boron diffusion, related isotope fractionation and the structural role of B in pegmatite forming melts

In this study, we experimentally investigate the diffusion of B in flux-rich (1.7 % Li2O, 2.5 % B2O3, 2.7 % P2O5 and 3.5 % F) pegmatite forming melts in order to assess the transport mechanisms of B in rare-element pegmatite deposits. Glasses were synthesized with either different B isotope compositions or with different B concentrations (up to 2.45 wt% B2O3). In order to explore the effect of water on transport properties, nominally dry and hydrous glasses (3.1–3.9 wt% H2O) were investigated. The produced glasses were combined to diffusion couples to study self-diffusion and chemical diffusion of B in the same chemical system. Experiments were conducted using an internally heated pressure vessel and a rapid-heat and rapid-quench cold-seal pressure vessel at 100 MPa Ar pressure in the temperature range of 850–1250 °C for 1.6–97.3 h. In chemical diffusion experiments, the flow of B is compensated by a combined opposing flow of all other cations and F, without changing their element ratios. Thus, effective binary diffusion coefficients are obtained. Similar activation energies were determined for B self-diffusion and chemical diffusion under nominally dry conditions (196 ± 6 kJ/mol and 200 ± 17kJ/mol, respectively). Many hydrous experiments show a tilted to strongly deformed interface after the run, which was probably formed by advective flow of the low-viscosity melts during heating under pressure. Based on the successful experiments, an activation energy of 138 ± 8 kJ/mol was estimated for chemical diffusion of B in hydrous melts. We show that B diffusivity correlates with the Eyring diffusivity and, therefore, with the melt viscosity. The stable isotopes of B fractionate kinetically along the diffusion profiles due to the faster diffusivity of 10B over 11B. This isotope fractionation can be quantified with an empirical isotope fractionation factor (β) of 0.032 ± 0.002. This effect is small, but significant and was previously not observed in experimental studies. While it is insensitive to the water contents used in our study, it seems to be strongly dependent on the experimental boundary conditions, such as the ratios of B between the diffusion couple halves and the B enrichment in the melt. Solid-state NMR experiments reveal that the majority of B is in trigonal coordination in our melts, which effectively weakens the melt structure. This further suggests that coordination differences are unlikely to be the driving force for B isotope fractionation between melt and fluid during late-stage fluid exsolution in pegmatitic systems.

ARTHROSPIRA-C space flight experiment: Validation of biomass and oxygen production bioprocesses using ground model demonstrator system

The Micro-Ecological Life Support System Alternative (MELiSSA) project of the European Space Agency develops a biological recycling loop for manned long-term space flight. The air revitalisation and food production are carried out by vascular plants and a photobioreactor containing the cyanobacterium Limnospira indica. In the space flight experiment ARTHROSPIRA-C, cultures of L. indica are run in a one-week batch mode (45 μmol photons m-2 s-1) followed by four semi-continuous cycles of two weeks length. Each cycle has a different, predefined light intensity following an increasing regime (45-55-70-80 μmol photons m-2 s-1). In this study, two ground science verification tests (SVT and δ-SVT) were conducted in a laboratory setting to test the reliability and functionality of the hardware- and software of ARTHROSPIRA-C. The SVT explored all four cycles and light intensities and δ-SVT was an additional test where only cycle 1 and 2 were performed. δ-SVT was used to investigate anomalies during SVT. These experiments revealed oxygen production rates between 0.10 ± 0.03 and 0.45 ± 0.01 mmol O2 L-1 h-1 and biomass production rates between 0.008 ± 0.000 and 0.021 ± 0.002 g L-1 h-1 while demonstrating sustained photosynthetic activity at all tested light intensities. In addition, proteomics analysis revealed interesting light intensity-induced effects on multiple pathways, whereas the lipidomic analysis reported no alterations. This study delves into the ground tests conducted during ARTHROSPIRA-C, paving the way for a forthcoming successful flight experiment scheduled aboard the International Space Station in autumn 2024.

Highly selective and sensitive N-amidothiourea-based fluorescence chemosensor for detecting Zn2+ ions and cell Imaging: Potential applications for plasma membrane detection

A “turn-on” fluorescent sensor L was developed for the selective detection of Zn2+ ions in biological settings. This sensor exhibited a specific response to Zn2+ ions, with a detection limit of 39 nM. Analyses through titration and Job’s plot confirmed that the formed complex has a 2:1 stoichiometry (L: Zn2+). The sensor displayed strong selectivity for Zn2+ over other metal ions, demonstrating a robust binding affinity while effectively reducing interference from heavy metals. The identification process was validated by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H NMR) titrations, mass spectrometry (MS), and Density Functional Theory (DFT) calculations. Maximum fluorescence intensity was achieved upon interaction with Zn2+ ions within a physiological pH range of 7.0–7.5. Moreover, successful imaging experiments in HEK293 cells highlighted the sensor’s potential as a powerful tool for monitoring interactions at the plasma membrane.

Probing shocks interacting with radiation waves with the Radishock experiment

Both radiation flows and shocks have been extensively studied in the laboratory in the past few decades due to their critical roles in many astrophysical and high-energy density physics processes. In the Radishock experiment, a halfraum-powered radiation wave is driven into a low-density foam and interacts with an ablatively driven, counter-propagating shock. The interacting waves produce a spike in energy density with a temperature greater than the local temperature of the individual waves. As in the successful predecessor experiment, COAX, the primary diagnostic uses absorption spectroscopy at many locations down the cylindrical target, enabling a spatial temperature inference of the radiation wave and its interactions with the shock. Combined with a radiography diagnostic that is capable of imaging the shock and interaction features, we are able to study and inform model predictions of the interaction spike phenomenon. We describe the underlying physics behind the shock interactions with the radiation front and the implications of this experimental study for a broad range of astrophysical phenomena.

Micropropagation of Selected Hybrid Tomato (Solanum lycopersicum L.) Varieties Using Shoot Tip Culture

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world. The use of hybrid varieties is a great option to increase its production but using F2 seed leads to lack of uniformity as a result of segregation. Therefore mass propagation using tissue culture could help to solve these problems. The objective of this study was to develop an optimum micropropagation protocol for Valouro, Uwezo and Shelter hybrid tomato varieties using shoot tip culture. Three successive experiments: shoot initiation; shoot multiplication and root inductions were conducted. Different concentrations of BAP (0.0, 0.25, 0.5, 0.75, 1.0 and 1.25 mg/l) and (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 mg/l) were used for shoot initiation and shoot multiplication, respectively. While different concentrations of IBA (0.0, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg/l) were used for root induction. The three separate experiments were laid out as factorial arranged in completely randomized design. The result of shoot initiation showed that the interaction of BAP*Varieties highly significant (P<0.01) effect. In the multiplication experiment, all parameters showed highly significant difference (P<0.01). In rooting experiment the interaction effect of IBA*Variety showed highly significant difference for the studied parameters. In conclusion MS medium without growth regulators was the optimum for shoot initiation. For shoot multiplication experiment 2mg/l, 0.5mg/l and 1.5mg/l of BAP was the optimum concentration for number of shoots per explant, shoot length, and leaf number respectively. For in vitro rooting MS medium containing 0.5 mg/l IBA was optimum for all varieties.

Design and Synthesis of Sulfur-Containing Tetradentate Ligands for Ir-Catalyzed Asymmetric Hydrogenation of Ketones.

Developing highly active and enantioselective ligands for the asymmetric hydrogenation of ketones has consistently attracted significant attention from scientists. A series of novel tetradentate sulfur-containing ligands, termed as f-thiophamidol, were successfully designed and synthesized which exhibited excellent performance in the asymmetric hydrogenation of simple ketones (up to 99% yield, 99% ee) and α-substituted β-keto sulfonamides (up to 99% yield, 99% ee, 99:1 dr). The subsequent successful gram-scale experiments with high TON demonstrated the immense potential application value of this system in synthesizing drug molecules.

Evaluating the efficacy of water alternating gas injection technique in the upper jurassic hydrocarbon reservoirs, considering saturation pressure

A tertiary method of enhanced oil recovery (EOR) based on the watergas alternating injection process (WG) is examined in this article based on the ratio between saturation pressure and reservoir pressure. A hydrodynamic simulation of the WAG process was carried out using TEMPEST MORE software (version 7,1). Many hydrocarbon fields worldwide are currently experiencing declining production rates. However, by utilizing advanced technologies like alternate injection of water and associated petroleum gas, the final oil recovery factor (RF) can be significantly increased. To ensure successful water injection experiments in real fields, it is crucial to consider the ratio of saturation and reservoir pressures during injection. Any negligence in this regard can lead to failure of the experiments aimed at enhancing oil recovery through WAG application. The research examines two variations of reservoir system models with certainty. In the initial model, the saturation pressure is lower than the reservoir pressure, signifying an under-saturated state of the reservoir system containing dissolved gas. Consequently, when accompanying petroleum gas is introduced, it mixes with oil at precise thermobaric conditions. According to the second model, the reservoir is saturated with gas at a saturation pressure equivalent to the formation pressure. A thorough assessment indicates that the success of water alternating gas injection (WAG) is heavily influenced by the initial state of the reservoir. In situations where the reservoir is already saturated with gas, implementing water-gas stimulation may be ineffective as gas breakthroughs may occur rapidly. However, implementing WAG in saturated reservoirs can lead to a 1 – 8% rise in overall oil production, observable within 3 – 4 years, depending on the timing after waterflooding. Simultaneously, in the case of reservoirs with a low saturation pressure, which exhibits an undersaturated reservoir system, the implementation of water alternating gas injection can be seen as a viable approach for enhancing oil recovery. WAG is a proven method that confidently increases oil recovery up to 10% in undersaturated reservoirs compared to conventional waterflooding.

Research Status of Sarcosaprophagous Beetles as Forensic Indicators.

In forensic entomology, research focused on sarcosaprophagous flies, but the sarcosaprophagous beetles, as important "forensic indicator species" of late-stage PMI in cadaver decomposition, received less attention. To increase attention on, and use and understanding of, sarcosaprophagous beetles in forensic entomology, this paper presents a bibliometric analysis of the available relevant literature. The occurrence frequency of beetle families and species from succession studies, actual cases, and experiments were calculated and illustrated using graphs. As a result, a total of 14 families and 1077 species associated with carcasses were collected, with Staphylinidae being the most frequently recorded among the families, and Necrobia rufipes (Fabricius, 1781) (Coleoptera: Cleridae) being the most frequently recorded species. In addition, a brief introduction of the cadaver-related beetles of each family is given, and research on the species identification of the immature stages, age estimation of the immature stages, and estimation of the arrival time of sarcosaprophagous beetles are discussed and prospected. This work will aid in the increased use of sarcosaprophagous beetles in forensic science practice in the future.

Nuclear-excited source of coherent and incoherent radiation with direct nuclear pumping

Uranium fission fragments, as well as the products of 3He(n,p)3H and 10B(n,α)7Li nuclear reactions were utilized in the nuclear reactor for gas ionization and excitation. However, the 6Li(n,α)3H nuclear reaction was less examined. The use of lithium-6 as a surface source of excitation of the gas medium, due to the long path length of tritium nuclei in the gas, allows to excite large volumes of gas as opposed to using 235U or 10B.While investigating the luminescence of noble gases in the core of the IVG.1M research reactor, we noted an appearance of alkali metal lines and a sharp increase in the intensity of these lines at temperatures above 570 K. It was determined that the population of levels of lithium atoms has practically no effect on the population of the 2p-levels of atoms of noble gases. The selectivity of p- and s-levels deactivation by lithium atoms implies the possibility of creating inversion of population at 2p-1s transitions of noble gas atoms. Successful experiments to study the luminescence of gases upon excitation by 6Li(n,α)3H nuclear reaction products allow us to proceed to experiments to achieve the laser action threshold and study the lasing characteristics of gas mixtures at the IGR pulsed nuclear reactor with thermal neutron flux density up to 7∙1016 n/cm2s. For this purpose, an experimental device designs were proposed to perform experiments on the IGR reactor. A step-by-step procedure of fabrication of a nuclear-excited source for excitation of gas mixtures is provided. The results of reactor experiments aimed at determining the spectral and temporal characteristics of optical radiation during excitation of gas mixtures by 6Li(n,α)3H nuclear reaction products are presented.

Enhancing concrete structure maintenance through automated crack detection: A computer vision approach

This paper presents the development of an Artificial Intelligence (AI) and Machine Learning (ML) model designed to detect cracks on concrete surfaces. The objective is to enhance the automation, precision, and performance of crack detection using the computer vision algorithm. Employing a ML approach and the YOLOv9 algorithm, this study developed a system to accurately identify concrete cracks from a diverse dataset. A total of 16,301 images of concrete surfaces, balanced between those with and without cracks, were utilized. The dataset was split into various sets with different ratios to ensure comprehensive model training. A transfer-learning methodology was employed to optimize the model's performance. The accuracy of the model was measured in each experiment to determine the optimal result. The most successful experiment resulted in a model with a mean Average Precision (mAP) of 94.6%, a Precision of 94.1%, and a Recall of 88.4%. These results demonstrate the effectiveness of AI and ML in concrete crack detection.

Investigating Pulmonary Neutrophil Responses to Inflammation in Mice via Flow Cytometry.

Neutrophils play a crucial role in maintaining lung health by defending against infections and participating in inflammation processes. Here we describe a detailed protocol for evaluating pulmonary neutrophil phenotype using a murine model of sterile inflammation induced by the fungal cell wall particle zymosan. We provide step-by-step instructions for the isolation of single cells from both lung tissues and airspaces, followed by comprehensive staining techniques for both cell surface markers and intracellular components. This protocol facilitates the sorting and detailed characterization of lung neutrophils via flow cytometry, making it suitable for downstream applications such as mRNA extraction, single-cell sequencing, and analysis of neutrophil heterogeneity. We also identify and discuss essential considerations for conducting successful neutrophil flow cytometry experiments. This work is aimed at researchers exploring the intricate functions of neutrophils in the lung under physiological and pathological conditions with the aid of flow cytometry.