Fluorescent Labeling Research Articles

P2Y12 receptor ligands enable design of fluorescent imaging probes for microglia

Abstract Microglia, the resident macrophages of the brain, have recently been revealed to be essential in regulating brain functions. However, there are limited methods that allow fluorescence labeling of microglia for their functional analysis. Herein, we demonstrate that a ligand for P2Y12 receptors, which is specifically expressed in microglia in the brain, can be leveraged for rational design of microglia imaging probes. Our design strategy is flexible as it can tune the fluorescence wavelength, affording new fluorescent probes of microglia.

Harnessing hetero-atom doped CQDs from Pyrostegia venusta for latent fingerprint and anti-counterfeit applications

The development of innovative techniques for latent fingerprint (LFP) analysis and anti-counterfeiting applications is crucial for addressing various societal challenges. Quantum dot (QD)-based fluorescent staining offers a promising approach due to its high sensitivity. However, the toxicity of traditional heavy metal-containing QDs necessitates the exploration of eco-friendly alternatives. This study presents a facile and environmentally sustainable method for synthesizing heteroatom (nitrogen, phosphorus, and potassium)-doped carbon quantum dots (N-P-K@CQDs) derived from the flower extract of Pyrostegia venusta. Characterization revealed spherical CQDs with an average size of approximately 4.7 nm, a quantum yield of 25 %, and excellent water solubility, photostability, and greenish-fluorescent properties. N-P-K@CQDs were incorporated into a cornstarch nanocomposite phosphor, which exhibited greenish fluorescence under UV excitation. The nanocomposite was utilized as a fluorescent marker for LFP analysis using a customized smartphone-based dactyloscope. Python-based image processing algorithms were employed to enhance and analyze the fluorescence images of the developed LFPs. Additionally; a proof-of-concept anti-counterfeiting tag was demonstrated using CQDs-based ink, enabling the detection of concealed security marks. The findings highlight the potential of eco-friendly N-P-K@CQDs as a versatile platform for both LFP analysis and anti-counterfeiting applications.

Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

Analysis of immunopathogenesis of psoriasis in the dynamics of treatment

Оbjective — to analyze the parameters of the immune system of the organism in patients with psoriasis before and after treatment. Materials and methods. The parameters of cellular immunity in an organism estimated after the change of relative and absolute amount of T- (CD3+) and B- (CD19+) of lymphocytes and subpopulations (CD4+, CD8+, CD16+, CD25+, CD71+, CD30+, CD95+). To identify of surface structures of lymphocytes the method of direct immune fluorescence was used where fluorescent label was attached to anti-CD of mononuclear antibodies of series Leu of the firm «Becton Dikinson» (USA). Calculations were conducted on the laser instantaneous cytofluorometer of the firm «Baston» (USA). Results and discussions. The most positive influence on the parameters of the cellular link of the immune system of the organism was found in patients with psoriasis who received immunobiological therapy with the medication etanercept in combination with narrowband (311 nm) phototherapy. After treatment in patients of this group, due to a significant decrease in the production of TNF and its serum concentration, the pro-inflammatory changes in the cellular link of the immune system decreased, the number of lymphocytes with the an early activation marker, which initiated a further cytokine cascade of development and prolongation of inflammation decreased significantly. In addition, the established reduction in the number of CD30+ lymphocytes is an indirect factor indicating the switching of the Tx2 response (autoimmune manifestations) to Tx1, and hence the achievement of the clinical and immunological remission of the psoriatic process. It is extremely important for the functioning of the immune system the decrease in the content of activated lymphocytes that express the Fas-receptor as due to the increased lymphocyte apoptosis, namely, T-cytotoxic lymphocytes, and autoimmune and proliferative changes took place inherent in the exacerbation of the psoriatic process. Conclusions. It was established that all our schemes of psoriasis treatment have immunorehabiting properties but their degree of severity was different. The most effective treatment scheme is the combined use of etanercept and UVB therapy, thus proliferative and inflammatory changes in the skin reduced, antigenic loading decreased, the level of CICs, autosensibilization and autoimmune disorders also decreased.

Robust Natural Light-Absorbable and -Degradable AIE Photosensitizers for Fluorescence Labeling and Efficient Photodynamic Eradication of Algal Pollutants.

Current water pollution caused by the excessive proliferation of harmful algae urges green methods that can efficiently utilize natural light to treat algal pollution. Herein, a series of aggregation-induced emission (AIE) photosensitizers that can efficiently harness sunshine were synthesized for the environmentally friendly and biocompatible treatment of algal pollution. By tuning the number of thiophene units and the electron conjugation degree, the photosensitizers' absorptions were broadened to cover the whole visible light range. The positive charges guided photosensitizers to aggregate on algal cell surfaces, resulting in a turn-on fluorescence signal and robust reactive oxygen species generation under sunshine, thereby achieving fluorescence labeling and photodynamic eradication of algae. The eradication outcomes demonstrated that the AIE photosensitizers significantly outperformed the commercial algaecide ALG. At 20 ppm photosensitizers, 90.4% and 94.2% killing rates were achieved for C. reinhardtii and C. vulgaris, respectively, 2.8- and 3.6-fold higher than those from the same concentration of ALG. Excellent performances in inhibiting algae growth were also verified with efficiency superior to that of ALG. Importantly, the photosensitizers can self-degrade into biocompatible fragments under irradiation to avoid secondary pollution. The developed photosensitizers that possess sunshine convertibility and degradability provide an efficient tool for algal treatment, showing broad research and application prospects.

Labeled and Label-Free Target Identifications of Natural Products.

Target identification, employing chemical proteomics, constitutes a continuous challenging endeavor in the drug development of natural products (NPs). Understanding their targets is crucial for deciphering their mechanisms and developing potential probes or drugs. Identifications fall into two main categories: labeled and label-free techniques. Labeled methods use the molecules tagged with markers such as biotin or fluorescent labels to easily detect interactions with target proteins. Thorough structure-activity relationships are essential before labeling to avoid changes in the biological activity or binding specificity. In contrast, label-free technologies identify target proteins without modifying natural products, relying on changes in the stability, thermal properties, or precipitation in the presence or absence of these products. Each approach has its advantages and disadvantages, offering a comprehensive understanding of the mechanisms and therapeutic potential of the NPs. Here, we summarize target identification techniques for natural molecules, highlight case studies of notable NPs, and explore future applications and directions.

Fluorescent labeling of live-cell surfaceome and its application in antibody-target interaction analysis

BackgroundCell-surface proteins play key roles in the communication between external stimuli and internal signaling. As protein types and expression levels vary in different cells, in-situ visualization of the whole surface proteome (surfaceome) may facilitate the study of their functions in homeostasis maintenance or response to environmental changes (e.g., drug treatment). However, there lacks easily-prepared and universal labeling probes to visualize them in living cells. ResultsWe designed and synthesized a small-molecule fluorescent probe, SRB-NHS, for one-step labeling of surfaceome. Live-cell imaging results exhibited the plasma membrane localization of the fluorescent signal from SRB-NHS and SDS-PAGE/fluorescence scanning results confirmed the covalent labeling of proteins by SRB-NHS, indicating the suitability of SRB-NHS for surfaceome labeling towards different cell lines. SignificanceUpon labeling by SRB-NHS, the cellular internalization of surfaceome was studied under different stimuli (e.g., nutritional deprivation, drug treatments). Intriguingly, specific monitoring of the interaction between antibody drugs and related cell-surface targets can be achieved when the probe is used in combination with fluorescently labeled antibodies and imaged via Förster resonance energy transfer (FRET), offering a new method compatible with various cell lines to monitor the surfaceome or a specific drug-target interaction in situ.

Tyramine-assisted fluorescent labeling of polysaccharides: Characterization, and interfacial properties

Fluorescence labeling has been widely used in various fields, including fluorescent sensors, biochemistry, medical and chemical research. This study proposed an efficient strategy for the detection and microanalysis of polysaccharides. Soy hull polysaccharide (SHP) was successfully labeled with fluorescein isothiocyanate (FITC) via an amination reaction using tyramine as a linker. The labeled polysaccharide (FTSHP) was characterized by fluorescence, UV–visible, flourier transform infrared (FT-IR), 1H NMR, differential scanning calorimetry (DSC) and interfacial tension. The results indicated that the labeling efficiency of FTSHP with different concentration of FITC (0.20–0.35 wt%) was 1.51 %, 1.58 %, 1.63 %, and 1.67 %, respectively, with fluorescence intensity increasing as FITC concentration increased. Moreover, the interfacial adsorption capacity and thermal stability of FTSHP were significantly improved compared to SHP. This labeling method offers a promising approach for detection and analysis of polysaccharides, providing new insights into the study of other natural polysaccharides.

Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

Nanoparticle Tracking Analysis: An Effective Tool to Characterize Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-enclosed particles that have attracted much attention for their potential in disease diagnosis and therapy. However, the clinical translation is limited by the dosing consistency due to their heterogeneity. Among various characterization techniques, nanoparticle tracking analysis (NTA) offers distinct benefits for EV characterization. In this review, we will discuss the NTA technique with a focus on factors affecting the results; then, we will review the two modes of the NTA techniques along with suitable applications in specific areas of EV studies. EVs are typically characterized by their size, size distribution, concentration, protein markers, and RNA cargos. The light-scattering mode of NTA offers accurate size, size distribution, and concentration information in solution, which is useful for comparing EV isolation methods, storage conditions, and EV secretion conditions. In contrast, fluorescent mode of NTA allows differentiating EV subgroups based on specific markers. The success of fluorescence NTA heavily relies on fluorescent tags (e.g., types of dyes and labeling methods). When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids, such as saliva, blood, and serum. Finally, we will discuss the limitations and future directions of the NTA technique in EV characterization.

Structural characterization of green fluorescent protein in the I-state

Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.

In vitro spectroscopic studies of 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride with main carrier plasma proteins

Current methods of cancer treatment, particularly chemotherapy, are associated with harmful side effects. For this reason, it is significant to study new substances with anticancer potential with the highest possible efficacy and the lowest possible side effects. The aim of the study was the spectroscopic analysis of the interaction between 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride (Salt3) and main carrier proteins, such as human serum albumin (HSA), α1 acid glycoprotein (AGP), human γ globulin (HGG) and controlled normal serum (CNS).The association constants (Ka [mol·L-1]) and the number of binding site classes (n) for the binding of Salt3 with studied carrier proteins and controlled normal serum were calculated using the Klotz equation. To study HSA and AGP high affinity binding sites, the fluorescent markers were used. Spectral parameter A and the second derivative of differential absorption spectra were used to assess environmental changes around aromatic amino acids residues. The changes in HSA and AGP secondary structure in the complexes with Salt3 were evaluated using the analysis using circular dichroism.Salt3 slightly binds to HSA, AGP, HGG molecules and CNS. In addition, Salt3 affects the tertiary structure of the studied proteins, while it does not damage the secondary structure of the main carrier proteins responsible for Salt3 distribution in the bloodstream.Because Salt3 binds weakly to model carrier proteins and normal control serum, it can lead to both strong therapeutic and toxic effects. Considering these preliminary spectroscopic studies, additional tests as well as expanding research to include other techniques seem justified.

Study on the Recombinant Human Interferon α1b, α2b, and Gamma Transient Expression and in Vitro Activities in Tobacco.

Interferons (IFNs) are universally acknowledged for their pivotal role in antiviral and anticancer responses. Thus, the primary aim of our study was to explore the expressions of IFN-α1b, α2b, and gamma in tobacco leaves via agrobacterium-mediated transient transformation and investigate their possible activities. Briefly, fusion with green fluorescent protein tags aided in detecting the expressed IFN proteins in the foliar tissues. The genetic constructs encoding these fusion proteins were inserted into the MagnICON plant transient expression vector, followed by transformation into the Agrobacterium strain GV3101. The transformed bacteria were then used to infiltrate tobacco leaves. After post-infiltration, protein expression was confirmed within 72 h via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the fusion proteins were subsequently purified using high-performance liquid chromatography for identification. Both the antiviral and anticancer potencies of these IFN fusion proteins were evaluated using the WISH/VSV (WISH cells/Vesicular stomatitis virus) microneutralization and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. Results indicated robust expression of the targeted IFN genes in plant tissues and significant biological activities against pathogens and cancer cells. Consequently, this study substantiated the viability of producing these therapeutic proteins in plants, potentially revolutionizing the manufacture of interferons biologically.

Paeoniflorin regulates microglia-astrocyte crosstalk, inhibits inflammatory response, and alleviates neuropathic pain through HSP90AA1/HMGB1 signaling pathway

Given the unclear, complex pathogenesis of neuropathic pain and the potential of paeoniflorin in relieving neuropathic pain, this study aimed to further clarify the therapeutic effect of paeoniflorin on neuropathic pain and to preliminarily explore the possible protective mechanisms of paeoniflorin. Chronic constrictive injury-induced Sprague Dawley rats and lipopolysaccharide-induced BV-2 cells were used for in vivo and in vitro experiments, respectively. The exosome uptake assay of mouse astrocytes (PKH-67 fluorescent labeling) and the mechanical nociceptive assay (the von Frey fibrous filaments) were performed. The effects of paeoniflorin and its downstream mechanisms on microglial and astrocyte activation, inflammation-associated proteins and exosome marker were determined. Paeoniflorin alleviated mechanical abnormal pain, decreased levels of ionized calcium binding adapter molecule-1 (Iba-1), glial fibrillary acidic protein, Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1, inflammatory factor) and High Mobility Group Box 1 (HMGB1, inflammation-related protein), and inhibited neuronal apoptosis in chronic constrictive injury rats or lipopolysaccharide-induced BV-2 cells. However, these effects were offset by HSP90AA1 overexpression in lipopolysaccharide-induced BV-2 cells. Exosomes of BV-2 cells could be absorbed by mouse astrocytes. In addition, HSP90AA1 overexpression reversed the effects of paeoniflorin on HMGB1 expression and inflammatory factors and proteins in mouse astrocytes co-cultured with exosome. Collectively, paeoniflorin alleviates neuropathic pain and inhibits inflammatory responses in chronic constrictive injury by modulating microglia-astrocyte crosstalk through HSP90AA1/HMGB1 pathways, which further evidences the potential of paeoniflorin in the treatment of neuropathic pain.

High-efficiency expression of alginate lyase in Pichia pastoris facilitated by Vitreoscilla hemoglobin

Vitreoscilla hemoglobin (VHb) can enhance the ability of recombinant strains to express heterologous proteins under low-oxygen conditions. However, its mechanism of action in the Pichia pastoris expression system remains unclear. In this study, three VHb construction strategies were designed to elucidate the mechanisms by which VHb promotes heterologous protein expression in P. pastoris. Notably, the co-expression pattern involving the sequential expression of the 102C300C gene followed by the Vgb gene significantly improved enzyme activity in the recombinant strain X33-102C300C-Vgb. The enzyme activity was 203.4 ± 0.57 U/mL at 180 h of fermentation in the 5-L system, which was 20.7 % higher than that of the starting strain X33-102C300C. Fluorescent labeling experiments revealed for the first time that a dual-transcription unit approach achieved superior VHb expression, indicating its potential for further development. Furthermore, transcriptomic and metabolomic analyses demonstrated that VHb enhances the growth of recombinant yeast colonies by improving respiration-related metabolism under low-oxygen conditions. This, in turn, alleviated the repression of the expression alcohol oxidase (AOX) at high methanol concentrations, resulting in increased alginate lyase activity. This study provides a theoretical foundation for improving the target protein expression in recombinant P. pastoris during high-density fermentation.

New 4-styrylcoumarin Derivatives as Potentials Fluorescent Labels for Biomolecules: Application in RNA-fish Probes

New 4-styrylcoumarin Derivatives as Potentials Fluorescent Labels for Biomolecules: Application in RNA-fish Probes

Live Cell Imaging Studies on Orange Emitting Thiazole-Pyridone Azo Fluorophore and Its Latent Fingerprints, Computational, Electrochemical Sensing for Dopamine Detection.

The fluorescent materials have sparked a lot of research interests due to their unique electronic, optical and chemical characteristics. Here, we are intended to present a simple and facile synthesis of novel orange emitting thiazole-pyridone fluorescent tag (TPFT) by a simple diazo coupling reaction and the structural elucidation was carried out by IR, NMR (1H and 13C), UV-Vis, photoluminescence and HR-MS spectrometry. The solvatochromic behaviour of the TPFT offered crucial information about the formation of hydrazone and azo tautomeric forms. The DFT simulations are computed to calculate HOMO-LUMO energy gap (3.028 eV) of TPFT along with MEP and RDG analyses. Comprehensive LFP visualization is revealed under both normal and UV light conditions (365 nm). The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to analyse the electrochemical behaviour of the TPFT-modified glassy carbon electrode (MGCE) and exhibited a lower detection limit of 7.89 × 10-8 M (S/N = 3) with a linear range of 0.5-8.0 μM for DA detection. The live-cell imaging study of TPFT showed a strong blue emission at 453 nm, which generally indicates the existence of fluorescence stability.

Injectability of high concentrated suspensions using model microparticles

Administration of high-concentrated suspension formulations (i.e., solid particles dispersed in a liquid vehicle) can be limited due to their greater propensity for needle occlusion. The physical interaction between the solid phase (i.e., particles), the vehicle (i.e., flow field), and injection devices could result in the formation of particle bridging or filtering, posing a major risk in dose delivery accuracy and injectability. Here, given the limited understanding on how clogging initiates in syringe and needle delivery systems, we report an experimental approach to fully characterize the transient injection behavior of suspensions. In particular, we first established a custom fluorescence tagging and imaging technique with integrated force sensor to enable visual observation of local particle concentrations and plunger force monitoring throughout injection. Then, we investigated the effects of key formulation properties and device parameters including particle concentration and morphology, carrier viscosity, injection rate, needle and syringe sizes, and tissue backpressure on the incidence of suspension particle jamming and needle clogging. We performed systematic benchmark studies demonstrating that increasing needle inner diameter (ID) and particle density considerably reduced clogging risk, while increasing vehicle viscosity, particle size, and tissue backpressure significantly increased clogging. The experimental framework presented is amenable to quantifying clogging risk in drug-loaded particle suspensions and provides a guideline to make informed decisions on the tradeoffs between creating particles for pharmaceutical impact and feasibility of injection delivery.

A fluorescence biosensor for organophosphorus pesticide detection with a portable fluorescence device-based smartphone

An innovative fluorescence biosensor was successfully developed to detect organophosphorus pesticide (OPs) by utilizing smartphone technology. The assay relied on the enzymatic activity of alkaline phosphatase (ALP), which facilitated the conversion of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAP) into L-ascorbic acid (AA). The AA that generated was then reactedwith o-phenylenediamine (OPD) to yield a fluorescent marker identified as 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxalin-1(3H)-one (DFQ). A novel bandpass approach was specifically developed for a smartphone that was integrated with a customized portable fluorescence device to measure the fluorescence emission of DFQ. The device has a unique application that converts the fluorescence intensity into an RGB signal. In the presence of OPs, malathion was chosen as the representative of the OPs substance; the enzymatic activity of the ALP was inhibited, resulting in a decrease in fluorescence intensity, which was proportional to the concentration of malathion. Smartphones can be used to measure fluorescence emission, offering a calibration sensitivity more than 70 times higher than that of conventional spectrofluorometer. The recently developed methodology can be employed to identify malathion within the concentration range of 0.1–1 ppm, with a detection limit of 0.05 ppm. The practical applicability of the method was established using vegetable samples, and the acquired results were in good agreement with those obtained using the standard HPLC approach. This innovative method provides both portability and accuracy, while also exhibiting a notable degree of sensitivity in detecting traceamounts of OPs.

Impact of membrane surface and module damage on virus removal and integrity in RO membranes

RO membrane modules are very effective in removing viruses and salts, but concerns remain regarding their long-term integrity in full-scale RO spiral wound systems. Membrane defects can arise from delamination, chlorine, or abrasive particle exposure, and module defects can arise from permeate tube, O-ring, and glue line damage. These issues compromise the membrane and module's performance, allowing viruses to pass that are not detected by conductivity monitoring. This paper assesses the impact of damage on virus removal using biological indicators (natural virus markers (NV)) and non-biological surrogates (salt, sulfate, rhodamine WT, pyranine). Three categories of module damage were studied and characterized using different autopsy techniques: membrane module component damage (permeate tube damage, O-ring damage, and membrane delamination), oxidative membrane damage (by hypochlorite exposure dose), and membrane surface damage (by abrasive particle exposure). Module component damage resulted in increased water permeability and decreased natural virus rejection. The conductivity remained similar to that of an intact module except in the case of permeate tube damage. Chlorine exposure (9000 ppm.h) didn't result in detectable NV markers in the permeate, indicating an intact support layer despite active layer damage. Abrasive particle exposure by suspended silicon carbide in the feed resulted in scratches, observed by SEM images, and a decline in the rejection of fluorescent marker, which indicates membrane surface damage. Results demonstrate that NV markers most consistently determine virus removal capacity in modules compromised by component damage. However, for assessing active layer damage, solutes like rhodamine-WT and pyranine prove more effective. This underscores the necessity of employing multiple indicators for a comprehensive evaluation of membrane integrity.