Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Spectrofluorometric determination of ascorbic acid in the plasma matrix: exploring correlation with autism spectrum disorder.

Ascorbic acid (Vitamin C) is crucial for bodily functions, including collagen synthesis, immune system support and antioxidant defense. Despite autism spectrum disorder's multifactorial nature involving genetic, environmental and neurological factors, robust evidence exploring the association between ascorbic acid and this disorder is notably lacking. This study introduces an innovative spectrofluorometric method to quantify ascorbic acid in the plasma of healthy children and those with autism spectrum disorder. The method relies on the interaction of ascorbic acid with the fluorescent dye propidium iodide. In acidic conditions, propidium iodide undergoes protonation and selectively binds to the negatively charged ascorbic acid forming an ion-pair complex. This complex alters the molecular structure of propidium iodide inducing chemical fluorescence quenching, that can be utilized for ascorbic acid quantification. The developed method undergoes rigorous validation following ICH guidelines, demonstrating a linear relationship within a concentration range of 4-40 µg/mL, with high precision and accuracy metrics. Analysis of real plasma samples from autistic and healthy children reveals clinically and statistically elevated levels of ascorbic acid in those with autism spectrum disorder.

Establishment of a rapid, cost-effective, and accurate method for assessing insect sperm viability.

High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. Sperm viability has been assessed by fluorescent staining of sperm cells. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR 14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.

Flow cytometry-based quantitative analysis of cellular protein expression in apoptosis subpopulations: A protocol.

Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

Comparison of plate counting with flow cytometry, using four different fluorescent dye techniques, for the enumeration of Bacillus cereus in milk.

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10-1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC2 dye, to determine the viable number of cells, it was found that the DiOC2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis.

Rapid determination of antibiotic susceptibility of clinical isolates of Escherichia coli by SYBR green I/Propidium iodide assay.

Infections caused by pathogenic Escherichia coli are a serious threat to human health, while conventional antibiotic susceptibility tests (AST) have a long turn-around time, and rapid antibiotic susceptibility methods are urgently needed to save lives in the clinic, reduce antibiotic misuse and prevent emergence of antibiotic-resistant bacteria. We optimized and validated the feasibility of a novel rapid AST based on SYBR Green I and Propidium Iodide (SGPI-AST) for E. coli drug susceptibility test. A total of 112 clinical isolates of E. coli were collected and four antibiotics (ceftriaxone, cefoxitin, imipenem, meropenem) were selected for testing. Bacterial survival rate of E. coli was remarkably linearly correlated with S value at different OD600 values. After optimizing the antibiotic concentrations, the sensitivity and specificity of SGPI-AST reached 100%/100%, 97.8%/100%, 100%/100% and 98.4%/99% for ceftriaxone, cefoxitin, imipenem and meropenem, respectively, and the corresponding concordances of the SGPI-AST with conventional AST were 1.000, 0.980, 1.000 and 0.979, respectively. The SGPI-AST can rapidly and accurately determine the susceptibility of E. coli clinical isolates to multiple antibiotics in 60 min, and has the potential to be applied to guide the precise selection of antibiotics for clinical management of infections caused by pathogenic E. coli.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

Inflicting, Monitoring, Visualizing, and Quantitating Various Sterile Membrane Damages and the Repair Response in Dictyostelium discoideum.

Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.

Mechanism of Cell-Killing Activity of Plantaricin LD1 Against Escherichia coli ATCC 25922.

Plantaricin LD1 was purified from a potential probiotic strain, Lactobacillus plantarum LD1 previously isolated from indigenous food, Dosa. In this study, we have performed a detailed mechanism of action of plantaricin LD1 against Escherichia coli ATCC 25922 considering Micrococcus luteus MTCC 106 as control. The plantaricin LD1 showed a minimum inhibitory concentration (MIC) of 34.57 µg/mL and a minimum bactericidal concentration (MBC) of 138.3 µg/mL against M. luteus MTCC 106, whereas MIC 69.15 µg/mL and MBC 276.6 µg/mL were found against E. coli ATCC 25922. The efflux of potassium ions, dissipation of membrane potential (∆ψ), and transmembrane pH gradient (∆pH) of plantaricin LD1-treated cells suggested the membrane-acting nature of plantaricin LD1. Plantaricin LD1 also caused degradation of the genomic DNA of the target strains tested. The cell killing was confirmed by staining with propidium iodide and visualized under light and electron microscopes. The bacteriocin-treated cells were found to be ruptured, swollen, and elongated. Thus, the findings indicate plantaricin LD1 kills E. coli ATCC 25922 by interacting with the cell membrane resulting in the efflux of intracellular contents and also causing degradation of nucleic acids leading to cell death.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Wortmannin Inhibits Cell Growth and Induces Apoptosis in Colorectal Cancer Cells by Suppressing the PI3K/AKT Pathway.

BACKGROUND: Colorectal cancer (CRC) remains a significant contributor to mortality, often exacerbated by metastasis and chemoresistance. Novel therapeutic strategies are imperative to enhance current treatments. The dysregulation of the PI3K/Akt signaling pathway is implicated in CRC progression. This study investigates the therapeutic potential of Wortmannin, combined with 5-fluorouracil (5-FU), to target the PI3K/Akt pathway in CRC. METHODS: Anti-migratory and antiproliferative effects were assessed through wound healing and MTT assays. Apoptosis and cell cycle alterations were evaluated using Annexin V/Propidium Iodide Apoptosis Assay. Wortmannin's impact on the oxidant/antioxidant equilibrium was examined via ROS, SOD, CAT, MDA, and T-SH levels. Downstream target genes of the PI3K/AKT pathway were analyzed at mRNA and protein levels using RTPCR and western blot, respectively. RESULTS: Wortmannin demonstrated a significant inhibitory effect on cell proliferation, modulating survivin, cyclinD1, PI3K, and p-Akt. The PI3K inhibitor attenuated migratory activity, inducing E-cadherin expression. Combined Wortmannin with 5-FU induced apoptosis, increasing cells in sub-G1 via elevated ROS levels. CONCLUSION: This study underscores Wortmannin's potential in inhibiting CRC cell growth and migration through PI3K/Akt pathway modulation. It also highlights its candidacy for further investigation as a promising therapeutic option in colorectal cancer treatment.

Multiparametric Analysis of Apoptosis by Flow Cytometry.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process. This approach to analyzing multiple apoptotic characteristics simultaneously yields far more information than single-parameter assays. While more informative than single-parameter assays, these multicolor methods can still be analyzed on relatively simple flow cytometers, making them widely accessible.

Confocal Imaging of Seeds.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

Abstract INTRODUCTION Citronellal (Cit) possesses antifungal activity and has possible implications for reactive oxygen species (ROS) generation in Candida albicans. In this study, the effects of Cit on ROS generation and the mechanisms by which Cit exerts anti-Candida effects were examined. METHODS A 2′,7′-dichlorodihydrofluorescein diacetate assay was used to assess oxidative damage. Cell necrosis was determined by flow cytometry after FITC-Annexin V staining. Mitochondrial function was studied based on mitochondrial potential, metabolic activity (MTT assay), and phenotypic susceptibility on a non-fermentable carbon source. Membrane intactness and DNA damage were estimated by a propidium iodide (PI) uptake assay and 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS ROS generation was enhanced in response to Cit, leading to necrosis (2%). Additional hallmarks of cell death in response to Cit, such as mitochondrial membrane depolarization and DNA damage, were also observed. Cit treatment resulted in dysfunctional mitochondria, as evidenced by poor labeling with the mitochondrial membrane potential-sensitive probe rhodamine B, reduced metabolic activity (61.5%), and inhibited growth on a non-fermentable carbon source. Furthermore, Cit induced DNA damage based on DAPI staining. These phenotypes were reinforced by RT-PCR showing differences in gene expression (30-60%) between control and Cit-treated cells. Finally, PI uptake in the presence of sodium azide confirmed non-intact membranes and suggested that Cit activity is independent of the energy status of the cell. CONCLUSIONS Cit possesses dual anticandidal mechanisms, including membrane-disruptive and oxidative damage. Taken together, our data demonstrated that cit could be used as a prominent antifungal drug.

Schistosoma mansoni cercariae experience influx of macromolecules during skin penetration

We have observed that when cercariae penetrate the skin of mice, there is influx into their tissues of Lucifer Yellow and certain labelled molecules of up to 20 kDa molecular weight. This observation was made using a variety of fluorescent membrane-impermeant compounds injected into the skin before the application of cercariae. This unexpected phenomenon was investigated further by transforming cercariae in vitro in the presence of the membrane-impermeant compounds and examining the distribution by microscopy. In schistosomula derived from this procedure, the nephridiopore and surface membrane were labelled while the pre- and post-acetabular glands were not labelled. The region associated with the oesophagus within the pharyngeal muscle clearly contained the fluorescent molecules, as did the region adjacent to the excretory tubules and the germinal mass. We used cercariae stained with carmine to aid identification of regions labelled with Lucifer Yellow. Although the mechanism of this influx is unclear, the observation is significant. From it, we can suggest an hypothesis that, during skin penetration, exposure of internal tissues of the parasite to external macromolecules represents a novel host-parasite interface

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.