Fluorescence-based Analysis Research Articles

Factors to consider before choosing EV labeling method for fluorescence-based techniques.

A well-designed fluorescence-based analysis of extracellular vesicles (EV) can provide insights into the size, morphology, and biological function of EVs, which can be used in medical applications. Fluorescent nanoparticle tracking analysis with appropriate controls can provide reliable data for size and concentration measurements, while nanoscale flow cytometry is the most appropriate tool for characterizing molecular cargoes. Label selection is a crucial element in all fluorescence methods. The most comprehensive data can be obtained if several labeling approaches for a given marker are used, as they would provide complementary information about EV populations and interactions with the cells. In all EV-related experiments, the influence of lipoproteins and protein corona on the results should be considered. By reviewing and considering all the factors affecting EV labeling methods used in fluorescence-based techniques, we can assert that the data will provide as accurate as possible information about true EV biology and offer precise, clinically applicable information for future EV-based diagnostic or therapeutic applications.

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Immune dynamics in response to risk group 4 pathogens in nonhuman primate models

Abstract Despite the critical importance of emerging infectious disease research, the underlying immune responses to risk group 4 pathogens remain largely understudied relative to many other pathogens. Spectral flow cytometry has dramatically increased the capacity of fluorescence-based cell analyses, allowing for the development of comprehensive panels across a variety of cell types. We have developed a 33-color phenotypic panel that incorporates a plethora of markers to simultaneously identify numerous distinct cellular populations within circulation and in tissue with a deliberate and extensive characterization of monocytes, dendritic cells, and tissue-resident T cells. This panel utilizes multiple approaches to accurately identify appropriate cell markers that can be easily distinguished with minimal to no compensation, despite containing fluorochromes with similar excitation and emission spectra. Importantly, this high-complexity panel was implemented on both fresh and frozen samples of whole blood, peripheral blood mononuclear cells (PBMCs), and cells isolated from multiple peripheral tissues across several nonhuman primate (NHP) models and on frozen human PBMCs, making this a broad cross-reactive panel enabling direct comparisons among NHP species and with humans. This new phenotypic flow panel allows for broad insight into underlying immune responses to infections across species while remaining amenable to additional potential markers of interest.

Fluorescence-Based Image Analysis of Seepage Behavior in Drip Irrigation: Exploring Varied Fractal Grading in Media Permeability

In the recycling of low-value metallic elements, heap leaching is commonly employed. The particle size distribution is a crucial parameter in heap leaching implementation, and the percolation behavior of a heap has always been a focal point in heap leaching technology. This paper utilizes a novel form of ultraviolet fluorescence image acquisition and fluorescence image analysis to investigate the percolation process with different fractal dimension particle size distributions, where the maximum particle diameter is 10 mm. The ore used was low-grade copper ore. The results indicate that the new fluorescence image analysis method can reveal different percolation regions during the heap leaching process, aiding in a better understanding of heap leaching behavior. The combined study found that under irrigation conditions of 10 mL/min, seepage was more uniform under the heap structure formed by a particle gradation with a fractal dimension of 2.2.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Synthesis of bimetallic palladacycles via C[sbnd]H bond activation: Crystal structure, DNA binding and in vitro cytotoxicity study

Herein, we report the synthesis, characterization and in vitro anticancer activity of two μ-Cl and μ-O bridged dimeric cyclometalated palladium(II) complexes having general formula [Pd2(L1)Cl2] (1) and [Pd2(L2)Cl2] (2). The complexes are synthesized via palladium assisted aromatic CH bond activation of coordinating ligands (HL1/HL2) and thoroughly characterized by spectroscopic techniques. X-ray crystal structure analysis has confirmed the square planar geometry of the complexes. DFT and TDDFT results have interpreted the electronic structure and solution spectra of the complexes. Cytotoxicity of the complexes was studied by in vitro model with human gastric cancer cell lines (AGS). Cytotoxicity investigation reveals that the complexes have promising anti-cancer activity, with IC50 values of 13.03 µM and 17.86 µM for 1 and 2, respectively. UV–vis and fluorescence techniques are used to examine how the complexes interact with CT DNA. Fluorescence-based competitive binding analysis with ethidium bromide (EB) indicates that the complexes effectively displace EB from the EB-DNA complex.

Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria.

Cell-wall-less (L-form) bacteria exhibit morphological complexity and heterogeneity, complicating quantitative analysis of them under internal and external stimuli. Stable and efficient labeling is needed for the fluorescence-based quantitative cell analysis of L-forms during growth and proliferation. Here, we evaluated the expression of multiple fluorescent proteins (FPs) under different promoters in the Bacillus subtilis L-form strain LR2 using confocal microscopy and imaging flow cytometry. Among others, Pylb-derived NBP3510 showed a superior performance for inducing several FPs including EGFP and mKO2 in both the wild-type and L-form strains. Moreover, NBP3510 was also active in Escherichia coli and its L-form strain NC-7. Employing these established FP-labeled strains, we demonstrated distinct morphologies in the L-form bacteria in a quantitative manner. Given cell-wall-deficient bacteria are considered protocell and synthetic cell models, the generated cell lines in our work could be valuable for L-form-based research.

Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group’s high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Highly sensitive and selective ratiometric fluorescent and visual detection of Cu2+ based on the hydroxytyrosol–naphthoresorcin–quantum dots sensing platform

Fluorescence-based visual analysis has attracted considerable attention in on-site detection owing to their distinct color gradient changes, easy operation and high sensitivity. In this work, we construct an exceptionally simple dual-emission ratio fluorescence sensor through the synergy of hydroxytyrosol (HYT), naphthoresorcin (NR) and ZnCdSe/ZnS quantum dots (QDs), namely HYT–NR–QD, for the fluorescent and visual detection of Cu2+. The design of ratiometric nanosensor is based on our new finding that Cu2+ can catalyze the reaction between HYT and NR to produce a cyan-emitting fluorophore (emission: 480 nm), and the copper species existed in the form of Cu+ and Cu2+ in reaction system. After adding QDs (emission: 605 nm), the free Cu+, Cu2+ and the resulting fluorophore in this system caused a great reduction in the FL intensity of QDs, due to the rapid cation exchange reaction between QDs and Cu2+/Cu+ as well as the inner filter effect between QDs and the resulting fluorophore. Overall, Cu2+ induced emission intensity changes at 480 and 605 nm leads to a distinct color change from red to cyan. A ratiometric fluorescence Cu2+ sensor with a lowest concentration of 3.42 nM was developed. Furthermore, the concentration of Cu2+ can be conveniently reflected with a smartphone application by identifying the RGB value. Moreover, the method proposed by us can be explored for Cu2+ determination in environmental water samples with a satisfying result, providing a novel, simple-operating and on-site detection of Cu2+ in water samples.

DWH24: a new antibody for fluorescence-based cell death analysis

Antibodies have gained considerable importance in laboratory and clinical settings. Currently, antibodies are extensively employed for the diagnosis and treatment of several human diseases. Herein, using targeted and cell immunisation approaches, we developed and characterised an antibody clone, DWH24. We found that DWH24 is an IgM κ type antibody that enables excellent visualisation and quantification of dead cells using immunofluorescence, fluorescence microscopy, and flow cytometry. This property was proved by the spontaneous cell death of several tumour cell lines and stimulated T cells, as well as after chemo- and photodynamic therapy. Unlike conventional apoptosis and cell death markers, DWH24 binding occurred in a Ca2+- and protein-independent manner and enabled live imaging of cell death progress, as shown using time-lapse microscopy. The binding specificity of DWH24 was analysed using a human proteome microarray, which revealed a complex response profile with very high spot intensities against various proteins, such as tropomyosin variants and FAM131C. Accordingly, DWH24 can be employed as a suitable tool for the cost-effective and universal analysis of cell death using fluorescence imaging and flow cytometry.

Promoter G-Quadruplex Binding Styryl Benzothiazolium Derivative for Applications in Ligand Affinity Ranking and as Ethidium Bromide Substitute in Gel Staining.

Fluorescent compounds that can preferentially interact with certain nucleic acids are of great importance in new drug discovery in a multitude of functions including fluorescence-based displacement assays and gel staining. Here, we report the discovery of an orange emissive styryl-benzothiazolium derivative (compound 4) which interacts preferentially with Pu22 G-quadruplex DNA among a pool of nucleic acid structures containing G-quadruplex, duplex, and single-stranded DNA structures as well as RNA structures. Fluorescence-based binding analysis revealed that compound 4 interacts with Pu22 G-quadruplex DNA in a 1:1 DNA to ligand binding stoichiometry. The association constant (Ka) for this interaction was found to be 1.12 (±0.15) × 106 M-1. Circular dichroism studies showed that the binding of the probe does not cause changes in the overall parallel G-quadruplex conformation; however, signs of higher-order complex formation were seen in the form of exciton splitting in the chromophore absorption region. UV-visible spectroscopy studies confirmed the stacking nature of the interaction of the fluorescent probe with the G-quadruplex which was further complemented by heat capacity measurement studies. Finally, we have shown that this fluorescent probe can be used toward G-quadruplex-based fluorescence displacement assays for ligand affinity ranking and as a substitute for ethidium bromide in gel staining.

Beta-Caryophyllene Modifies Intracellular Lipid Composition in a Cell Model of Hepatic Steatosis by Acting through CB2 and PPAR Receptors

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-β-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.

Direct optical resolution in the intra-molecular distance range.

Estimations of molecular distances <10 nm have long been the almost exclusive domain of Förster resonance energy transfer (FRET) and related spectroscopic techniques. By site-specifically labeling molecular subunits with (at least) two fluorophores, single-molecule FRET has also allowed for the monitoring of distances within macromolecules. In this presentation, we report on the direct determination of such intra-molecular distances based on the MINFLUX localization concept. We measured intra-macromolecular spacings of photoactivatable small-molecule fluorophores in well-characterized uni- or few-molecular systems, with simple polypeptides and proteins serving as initial examples. Our experiments indicate that the widely employed STORM-type dark-state photoblinking of cyanines reaches an impasse for dye separations below ∼6-10 nm, as the dyes couple and no longer behave independently. The complex photophysics of such dye-dye communication impedes the reliable, isolated detection of their signals. As we show however, sequential photoactivation - providing the required off-on-off transitions in a more orthogonal way - is a viable strategy to read out dye coordinates even for separations as low as few nm, beyond any perceived remaining barriers of resolution. Our results, with direct resolution of intra-molecular distances and conformations, herald the possibility of fluorescence-based analyses of structural biology at room or physiological temperature relying on direct positional measurements rather than on indirect FRET readouts.

Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

Quantitative Fluorescence Analysis Reveals Dendrite-Specific Thalamocortical Plasticity in L5 Pyramidal Neurons during Learning.

High-throughput anatomic data can stimulate and constrain new hypotheses about how neural circuits change in response to experience. Here, we use fluorescence-based reagents for presynaptic and postsynaptic labeling to monitor changes in thalamocortical synapses onto different compartments of layer 5 (L5) pyramidal (Pyr) neurons in somatosensory (barrel) cortex from mixed-sex mice during whisker-dependent learning (Audette et al., 2019). Using axonal fills and molecular-genetic tags for synapse identification in fixed tissue from Rbp4-Cre transgenic mice, we found that thalamocortical synapses from the higher-order posterior medial thalamic nucleus showed rapid morphologic changes in both presynaptic and postsynaptic structures at the earliest stages of sensory association training. Detected increases in thalamocortical synaptic size were compartment specific, occurring selectively in the proximal dendrites onto L5 Pyr and not at inputs onto their apical tufts in L1. Both axonal and dendritic changes were transient, normalizing back to baseline as animals became expert in the task. Anatomical measurements were corroborated by electrophysiological recordings at different stages of training. Thus, fluorescence-based analysis of input- and target-specific synapses can reveal compartment-specific changes in synapse properties during learning.SIGNIFICANCE STATEMENT Synaptic changes underlie the cellular basis of learning, experience, and neurologic diseases. Neuroanatomical methods to assess synaptic plasticity can provide critical spatial information necessary for building models of neuronal computations during learning and experience but are technically and fiscally intensive. Here, we describe a confocal fluorescence microscopy-based analytical method to assess input, cell type, and dendritic location-specific synaptic plasticity in a sensory learning assay. Our method not only confirms prior electrophysiological measurements but allows us to predict functional strength of synapses in a pathway-specific manner. Our findings also indicate that changes in primary sensory cortices are transient, occurring during early learning. Fluorescence-based synapse identification can be an efficient and easily adopted approach to study synaptic changes in a variety of experimental paradigms.

Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA.

The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

Fluorescence-based visual analysis and inhibitor screening of Arylamine N-acetyltransferase 2, a key enzyme for tuberculosis

Tuberculosis is a global epidemic caused by pathogenic Mycobacterium tuberculosis (Mtb), which seriously threatens human life. Arylamine N-acetyltransferase 2 (NAT2) is a vital enzyme in the synthesis of Mtb wall, and closely related to the growth of Mtb. Herein, a fluorescence-based recognition tool (BDPN) has been developed for rapid, sensitive, and selective detection of NAT2 activity in Mtb. As well as a visual high-throughput screening approach for NAT2 inhibitors was established based on BDPN, and four active natural compounds derived from Psoraleae Fructus were discovered that exhibited strong inhibitory effect toward NAT2. Furthermore, Isobavachalcone exhibited the considerable anti-tuberculosis effect through prominently destroying the bacterial cell wall of Mtb and slow down its growth. Moreover, Isobavachalcone could delay the elimination of the anti-tuberculosis drug Isoniazid (INH) mediated by NAT2 and enhance anti-tuberculosis effect of INH. In summary, BDPN was a promising tool for monitoring endogenous NAT2 in Mtb and provided some useful guidance for the rational administration of anti-tuberculosis drug.

Inhibition of Plasmodium falciparum Hsp70-Hop partnership by 2-phenylthynesulfonamide.

Plasmodium falciparum Hsp70-1 (PfHsp70-1; PF3D7_0818900) and PfHsp90 (PF3D7_0708400) are essential cytosol localized chaperones of the malaria parasite. The two chaperones form a functional complex via the adaptor protein, Hsp90-Hsp70 organizing protein (PfHop [PF3D7_1434300]), which modulates the interaction of PfHsp70-1 and PfHsp90 through its tetracopeptide repeat (TPR) domains in a nucleotide-dependent fashion. On the other hand, PfHsp70-1 and PfHsp90 possess C-terminal EEVD and MEEVD motifs, respectively, which are crucial for their interaction with PfHop. By coordinating the cooperation of these two chaperones, PfHop plays an important role in the survival of the malaria parasite. 2-Phenylthynesulfonamide (PES) is a known anti-cancer agent whose mode of action is to inhibit Hsp70 function. In the current study, we explored the antiplasmodial activity of PES and investigated its capability to target the functions of PfHsp70-1 and its co-chaperone, PfHop. PES exhibited modest antiplasmodial activity (IC50 of 38.7 ± 0.7 µM). Furthermore, using surface plasmon resonance (SPR) analysis, we demonstrated that PES was capable of binding recombinant forms of both PfHsp70-1 and PfHop. Using limited proteolysis and intrinsic fluorescence-based analysis, we showed that PES induces conformational changes in PfHsp70-1 and PfHop. In addition, we demonstrated that PES inhibits the chaperone function of PfHsp70-1. Consequently, PES abrogated the association of the two proteins in vitro. Our study findings contribute to the growing efforts to expand the arsenal of potential antimalarial compounds in the wake of growing parasite resistance against currently used drugs.

Microporous affinity membranes and their incorporation into microfluidic devices for monitoring of therapeutic antibodies

Control of monoclonal antibody (mAb) concentrations in serum is important for maintaining the safety and efficacy of these lifesaving therapeutics. Point-of-care (POC) quantification of therapeutic mAbs could ensure that patients have effective mAb levels without compromising safety. This work uses mimotope-functionalized microporous alumina affinity membranes in vertical flow assays for detection and quantitation of therapeutic mAbs. Selective capture of bevacizumab from 1000:1 diluted serum or plasma and binding of a fluorescently labelled anti-human IgG secondary antibody enable fluorescence-based analysis of bevacizumab at its therapeutically relevant concentration range of ∼50–300 μg/mL. The assay results in a linear relationship between the fluorescence intensity of the antibody capture spot and the bevacizumab concentration. A simple prototype microfluidic device containing these membranes allows washing, reagent additions and visualization of signal within 15 min using a total of 5 mL of fluid. The prototype devices can monitor physiologically relevant bevacizumab levels in diluted serum, and future refinements might lead to a POC device for therapeutic drug monitoring.

Analysis of Peripheral Blood Basophils in Pediatric Systemic Lupus Erythematosus.

Basophils are the least abundant circulating leukocytes, and their immunological role has not yet been completely elucidated. There is evidence supporting their immunomodulatory role in several pathological settings; recently, studies in both experimental models and humans suggested that basophil homeostasis may be altered in systemic lupus erythematosus (SLE). Here, we first assessed circulating basophils in children affected with pediatric SLE (pSLE). In this cross-sectional study, circulating basophils were enumerated by fluorescence-based flow cytometry analysis in children affected with pSLE, in addition to children suffering from juvenile idiopathic arthritis (JIA) or non-inflammatory/non-rheumatic conditions. This study included 52 pediatric patients distributed in these three groups. We observed a statistically significant reduction of peripherally circulating basophils in children with pSLE compared to the other two groups of patients. This preliminary study is consistent with the available studies in adult patients with SLE showing a reduced number of circulating basophils. However, further research is needed to draw final conclusions on basophils’ homeostasis in pSLE, in addition to their correlation with the disease activity and concomitant therapies.