Alexa Fluor Research Articles

Polysaccharide-Based Coacervate Microgel Bearing Cationic Peptides That Achieve Dynamic Cell-Membrane Structure Alteration and Facile Cytosolic Infusion of IgGs.

Conjugates of the biocompatible polysaccharide pullulan with a cell membrane permeabilizing peptide L17E (PL-L17Es) were prepared with the aim of producing complex coacervates with pronounced intracellular antibody (IgG) delivery activity and stable structures. Coacervates with diameters of a few μm were formed simply by mixing PL-L17Es with IgG labeled with negatively charged fluorescent moieties of Alexa Fluor 488 [IgG(AF488)]. The coacervate resulted in a pronounced cytosolic infusion of IgG(AF488) and IgG binding to the target proteins inside the cell. The droplet structures were maintained even under high salt conditions, and the fluorescence in the droplet was not recovered after photobleaching, suggesting the formation of complex coacervate microgels. Dynamic changes in cell membrane structure to entrap the coacervate microgels were captured by confocal and electron microscopy, resulting in cytosolic IgG infusion. The use of M-lycotoxin instead of L17E resulted in a coacervate microgel with marked IgG delivery activity even in the presence of serum. Successful IgG delivery to primary hepatocytes, undifferentiated induced pluripotent stem (iPS) cells, and iPS cell-derived intestinal epithelial cells was also achieved. The construction of complex coacervate microgels with design flexibility and the validity of intracellular IgG delivery with high salt stability were thus demonstrated.

An Indirect Pathway from the Rat Interstitial Nucleus of Cajal to the Vestibulocerebellum Involved in Vertical Gaze Holding.

The neural network, including the interstitial nucleus of Cajal (INC), functions as an oculomotor neural integrator involved in the control of vertical gaze holding. Impairment of the vestibulocerebellum (VC), including the flocculus (FL), has been shown to affect vertical gaze holding, indicating that the INC cooperates with the VC in controlling this function. However, a network between the INC and VC has not been identified. In this study, we aimed to obtain anatomical evidence of a neural pathway from the INC to the VC (the INC-VC pathway) in rats. Injection of dextran-conjugated Alexa Fluor 488 or adeno-associated virus 2-retro (AAV2retro) expressing GFP into the FL or another VC region (uvula/nodulus) did not reveal any retrogradely labeled neurons in the INC, suggesting that INC neurons do not project directly to the VC. Rabies virus-based transsynaptic tracing experiments revealed that the INC-VC pathway is mediated via synaptic connections with the prepositus hypoglossi nucleus (PHN) and medial vestibular nucleus (MVN). The INC neurons in the INC-VC pathway were mainly localized bilaterally within the rostral region of the INC. Transsynaptic tracing experiments involving the INC-FL pathway revealed that INC neurons connected to the FL via the bilateral PHN and MVN. These results indicate that the INC-VC pathway is not a direct pathway but is mediated via the PHN and MVN. These findings can provide clues for understanding the network mechanisms responsible for vertical gaze holding.

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

Comparative fluorophotometric evaluation of the ocular surface retention time of cross-linked and linear hyaluronic acid ocular eye drops on healthy dogs.

Evaluate and compare the retention time on the canine ocular surface of crosslinked hyaluronic acid (X-HA), linear hyaluronic acid (L-HA) and saline solution using fluorescent compounds (fluorescein sodium salt, Alexa Fluor 488 cadaverine and Alexa Fluor 488 maleimide). 0.9% saline solution (SAL) was combined with fluorescein sodium salt. X-HA and L-HA were covalently modified using Alexa Fluor 488 reactive moieties. Eye drops were applied to 70 eyes of 35 dogs that were previously assessed and determined to have a normal ocular surface. Employing a blue light filter (450-490nm), digital images were captured from instillation to 180min. Images were analyzed to assess the percent of the total ocular area covered with green fluorescence at various time points. X-HA exhibited a dual phase behavior: A broad microgel coverage first, followed by accumulation in tear film meniscus and medial canthus in the second phase, remaining in contact with the ocular surface up to 180min. Coverage with L-HA and SAL eye drops quickly migrated to the tear meniscus. No traces of the fluorescent compounds were observed by 45min in eyes treated with SAL solution compound and, by 120min, eyes treated with L-HA. X-HA exhibited a significantly increased ocular surface contact time with the ocular surface compared with L-HA and SAL. Not only could this indicate extended lubrication time but also supports the potential use of this compound as a method for topical sustained-release drug application.

Effects of anti-rainbow trout germ-cell monoclonal antibody on germ cells and gonadal tissues in rainbow trout (Oncorhynchus mykiss)

The development of sterilization technology is important to improve the production efficacy of aquaculture and prevent genetically modified fish from escaping. Immunosterilization is a novel sterilization method that has already been used in mammals. In this study, anti-rainbow trout germ-cell monoclonal antibodies (anti-GC mAbs) were injected into immature rainbow trout Oncorhynchus mykiss, and their effects were analyzed. First, anti-GC mAbs labeled with Alexa Fluor 488 were injected into the body cavity of larvae 20 days (group 1) and 102 days (group 2) after fertilization. Second, anti-GC mAbs were injected into the body cavity of yearling trout (group 3), and their impacts on gonadal tissue were analyzed in terms of histology and gene expression levels. As a result, anti-GC mAbs were observed to bind to some primordial GCs in group 1, whereas there were no obvious positive cells in male testes of group 2. In contrast, green fluorescent signals were observed in an arrangement parallel to the ovarian lamina in female ovaries of group 2, which was similar to the arrangement of positive signals on ovarian tissue sections immunostained with anti-GC mAbs. GCs in group 1 were observed again after 76 days, and double-positive cells remained. In group 3, there were changes in the expression levels of GC-specific genes, but the changes were not seen as biologically meaningful. Moreover, there was no histological abnormality in gonadal tissues after immunization. In conclusion, these results suggested that anti-GC mAbs could only reach GCs in early developmental stages or in the ovary, and injection of anti-GCs mAbs did not have harmful effects on GCs after binding.

Retinal Input to Macaque Superior Colliculus Derives from Branching Axons Projecting to the Lateral Geniculate Nucleus.

The superior colliculus receives a direct projection from retinal ganglion cells. In primates, it remains unknown if the same ganglion cells also supply the lateral geniculate nucleus. To address this issue, a double-label experiment was performed in 2 male macaques. The animals fixated a target while injection sites were scouted in the superior colliculus by recording and stimulating with a tetrode. Once suitable sites were identified, cholera toxin - subunit B Alexa Fluor 488 was injected via an adjacent micropipette. In a subsequent acute experiment, cholera toxin subunit B - Alexa Fluor 555 was injected into the lateral geniculate nucleus at matching retinotopic locations. After a brief survival period, ganglion cells were examined in retinal flatmounts. The percentage of double-labeled cells varied locally, depending on the relative efficiency of retrograde transport by each tracer and the precision of retinotopic overlap of injection sites in each target nucleus. In counting boxes with extensive overlap, 76-98% of ganglion cells projecting to the superior colliculus were double-labeled. Cells projecting to the superior colliculus constituted 4.0 - 6.7% of the labeled ganglion cell population. In one particularly large zone, there were 5,746 cells labeled only by CTB-AF555, 561cells double-labeled by CTB-AF555 and CTB-AF488, but no cell labeled only by CTB-AF488. These data indicate that retinal input to the macaque superior colliculus arises from a collateral axonal branch supplied by about 5% of the ganglion cells that project to the lateral geniculate nucleus. Surprisingly, there exist no ganglion cells that project exclusively to the SC.Significance statement The retina contains a multitude of ganglion cell classes, projecting in parallel to different brain targets. The superior colliculus receives retinal input, but its source remains controversial. In two macaques, a green tracer was injected into the superior colliculus and an orange tracer into the lateral geniculate nucleus. When retinotopic overlap was optimal, ganglion cells containing the green tracer were also labeled by the orange tracer. This finding indicates that retinal input to the superior colliculus arises from axon collaterals of ganglion cells that project to the lateral geniculate nucleus, even though these two retinal targets serve utterly different functions. The next challenge is to determine the purpose of this projection and why it is shared.

Area-Efficient Mixed-Signal Time-to-Digital Converter Integration for Time-Resolved Photon Counting.

Digital histogram generation for time-resolved measurements with single-photon avalanche diode (SPAD) sensors requires the storage of many timestamp signals. This work presents a mixed-signal time-to-digital converter (TDC) that uses analog storage to achieve an area-efficient design that can be integrated in large SPAD arrays. Fabricated using a 150 nm CMOS process, the prototype occupies an area of only 18.3 µm × 36.5 µm, a notable size reduction compared to conventional designs. The experimental results demonstrated high performance, with an integral nonlinearity (INL) of 0.35/0.14 least significant bit (LSB) and a differential nonlinearity (DNL) of 0.14/-0.12 LSB. In addition, the proposed TDC can support the construction of histograms comprising up to 512 bins, making it an effective solution to accommodate a wide range of resolution requirements. Validated in a point-of-care (PoC) device for fluorescence lifetime measurements, it distinguished between lifetimes of approximately 4.1 ns, 3.6 ns and 80 ns with the Alexa Fluor (AF) 546 and 568 dyes and Quantum Dot (QD) 705, respectively. The analog storage design and area-efficient architecture offer a novel approach to integrating TDCs in SPAD-based systems, with potential applications in medical diagnostics and beyond.

Enhanced Performance of the Optimized Dye CF583R in Direct Stochastic Optical Reconstruction Microscopy of Active Zones in Drosophila Melanogaster.

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts.

Spectroscopic and solution properties characterization of quaternary ammonium ion containing polycations complexed with fluorescent rhodamine sulfonic acid dyes

Based on the growing range of applications for polycations in research and commercial materials, a continuing need exists to advance the fundamental knowledge and understanding of this class of materials. Spectroscopic and solution properties characterizations of noncovalently labeled, fluorescent Alexa Fluor® dye complexes of two commercial polycations, poly(2-(trimethylamino) ethyl methacrylate) monocation and poly[bis[2-chloroethyl] ether-alt-1,3-bis[3-(dimethylamino) propyl] urea] dication are reported to help address this need. A variety of fluorescence spectroscopic methods are used with a special emphasis on fluorescence correlation spectroscopy (FCS) which is applied to characterize the Stokes radius (RS) and equilibrium dissociation constants (Kd) of dye-polycation complexes at nanomolar dye concentrations. Resulting RS values indicate dye binding to individual polycation chains. Measured Kd values in the sub-micromolar range are consistent with strong dye binding. Increasing solution ionic strength with sodium chloride addition inhibits dye binding and decreases the RS of dye-polycation complexes due to size collapse of polycation chains. The complexes differ in their solution stability to ionic strength changes suggesting that both electrostatic and hydrophobic binding interactions influence dye binding. This study establishes the viability of noncovalent dye-polycation complexation in concert with FCS characterization as a general approach for investigating the properties of quaternary ammonium ion containing polycations in aqueous solution.

Long term investigation of formulation buffers to mitigate stability issues of conjugated critical reagents

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at −80 °C in PBS or a protein storage buffer for 3–15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

Avidin-positive mast cells of the liver when exposed to water-soluble silicon for nine months

Mast cells, due to the mediators contained in them, are active participants in various processes occurring in the body. The reaction of avidin-positive mast cells of the liver to the intake of silicon with drinking water at a concentration of 20 mg/L for nine months was studied. The experiment was conducted on laboratory nonlinear male rats, which were divided into two groups: the control group received bottled drinking water with a silicon concentration of 10 mg/L; the experimental group received the same water, but with the addition of sodium metasilicate nine-hydrate, which was used to adjust the total concentration of silicon in drinking water to 20 mg/L. The mass concentration of silicon in the water of the control and experimental groups was determined using an inductively coupled plasma emission spectrometer 5110 ICP-OES. After nine months, the animals were sacrificed, the liver was extracted and fixed in 10% neutral formalin, processed and embedded in paraffin. After deparaffinization, 6-ìm-thick liver sections were incubated for 30 minutes with green fluorescent-labeled avidin (Avidin, Alexa Fluor® 488 conjugate, Invitrogen, Germany). The preparations were analyzed under a fluorescence microscope at an excitation light wavelength of 495 nm. According to the results of the study, an increase in the number of avidin-positive mast cells due to weakly fluorescent ones was found in the liver of rats of the experimental group, as well as an increase in the median of their area and fluorescence intensity. It was revealed that the change in the median of area of avidin-positive mast cells in the liver of rats of the experimental group is due to a decrease in the number of small cells and an increase in the number of medium and large cells. Positive strong and positive average correlations were established between the cell area and its intensity in the control and experimental groups, respectively. Thus, the study made it possible to expand the data on the effect of water-soluble silicon on the liver and indirectly suggest the occurrence of inflammatory processes in the organ under study.

In vivo antibody labeling route and fluorophore dictate labeling efficiency, sensitivity, and longevity.

Leukocytes migrate through the blood and extravasate into organs to surveil the host for infection or cancer. Recently, we demonstrated that intravenous (IV) anti-CD45.2 antibody labeling allowed for precise tracking of leukocyte migration. However, the narrow labeling window can make this approach challenging for tracking rare migration events. Here, we show that altering antibody administration route and fluorophore can significantly extend the antibody active labeling time. We found that while both IV and intraperitoneal (IP) anti-CD45.2 antibody labeled circulating leukocytes after injection, they had different kinetic properties that impacted labeling time and intensity. Quantification of circulating antibody revealed that while unbound IV anti-CD45.2 antibody rapidly decreased, unbound IP anti-CD45.2 antibody increased over one hour. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 (AF647) and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared to other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not AF647, resulted in continuous blood leukocyte labeling for over 6 hours. Finally, we leveraged IP anti-CD45.2 BB700 antibody to track slower migrating leukocytes into tumors. We found that IP anti-CD45.2 antibody injection allowed for the identification of ~seven times as many tumor-specific CD8+ T cells that had recently migrated from blood into tumors. Our results demonstrate how different injection routes and fluorophores affect anti-CD45.2 antibody leukocyte labeling and highlight the utility of this approach for defining leukocyte migration in the context of homeostasis and cancer.

Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media.

Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638nm. An excitation power density of 0.60 kW/cm2 at 638nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.

Detection of exogenous siRNA inside sweet corn bundle sheath cells and the RNAi dynamics in the early stage of Maize dwarf mosaic virus infection

Maize dwarf mosaic virus (MDMV) is one of the most serious viruses of sweet corn. Utilising the process of RNA interference, the exogenous introduction of small RNA molecules mimicking virus-derived small interfering RNA (siRNA) into the plant prior to infection triggers the antiviral RNA silencing effect, thereby promoting more effective antiviral protection. Hence, a treatment with MDMV-derived small RNA was applied to sweet corn plants one day before MDMV virus inoculation. ALEXA FLUOR®488 fluorophore-bound exogenous siRNA was successfully detected inside intact sweet corn cells using confocal fluorescence microscopy. Furthermore, it was demonstrated that the exogenous siRNA treatment led to a notable upregulation of the AGO1, AGO2b, AGO10b, AGO18a, DCL1, DCL3a, DCL4, RDR1, and MOP1 genes within 24 h of the treatment. Overall, exogenous siRNA treatment resulted in better virus control of infected sweet corn plants, as indicated by the lower viral RNA and coat protein levels compared to the infected group without pre-treatment.

Plant-derived compounds normalize platelet bioenergetics and function in hyperglycemia

BackgroundPolyphenols have been shown to decrease oxidative stress and modulate glycemic response. Nevertheless, their effect on platelet bioenergetics and clot structure in diabetes and hyperglycemia is unknown. ObjectivesTo investigate the effect of polyphenols on human platelet bioenergetics and its subsequent effect on clot structure in normoglycemia vs acute hyperglycemia in vitro. MethodsFour polyphenols (resveratrol, hesperetin, epigallocatechin gallate [EGCG], and quercetin) were selected for initial analysis. Healthy volunteers’ isolated platelets/platelet-rich plasma were treated with 5 or 25 mM glucose to represent normoglycemia and acute hyperglycemia, respectively. Platelet-derived reactive oxygen species (ROS), citrate synthase activity (mitochondrial density), mitochondrial calcium flux, and mitochondrial respiration were performed following exposure to polyphenols (20 µM, 1 hour) to determine their effects on platelet bioenergetics. Procoagulant platelets (annexin V) and fibrin fiber density (Alexa Fluor-488 fibrinogen; Invitrogen) were analyzed by laser scanning confocal microscopy, while clot porosity was determined using platelet-rich plasma following exposure to polyphenols (20 µM, 20 minutes). ResultsAcute hyperglycemia increased ROS, mitochondrial calcium flux, maximal respiration, and procoagulant platelet number. Resveratrol, quercetin, and EGCG reduced platelet ROS in normoglycemic and acute hyperglycemic conditions. Mitochondrial density was decreased by quercetin and EGCG in normoglycemia. Resveratrol and EGCG reduced mitochondrial calcium flux in acute hyperglycemia. Resveratrol also decreased procoagulant platelet number and attenuated oxygen consumption rate in normoglycemia and acute hyperglycemia. No effect of hyperglycemia or polyphenols was observed on fibrin fiber density or clot pore size. ConclusionOur results suggest polyphenols attenuate increased platelet activity stemming from hyperglycemia and may benefit thrombosis-preventative strategies in patients with diabetes.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the β-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

Examination of Pyroptosis by Flow Cytometry.

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

A Dual Strategy-In Vitro and In Silico-To Evaluate Human Antitetanus mAbs Addressing Their Potential Protective Action on TeNT Endocytosis in Primary Rat Neuronal Cells.

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.

Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy

Blinking of fluorophores is essential in the context of single molecule localization-based optical super-resolution microscopy methods. To make the fluorescence molecule undergo blinking specific complex chemical mounting buffer systems, combined with suitable oxygen scavengers, and reducing agents are required. For instance to realise blinking in widely used fluorescence tags, like Alexa Fluor 647 (AF647), they are to be mounted on anti-fading buffer such as Mowiol and reducing agent such as Beta (β) - ME. However, the quality of the super-resolved images is decided by the total number of blinking events or in other words net duration for which the fluorescence blinking persists. In this paper we investigate how a violet and UV light induced fluorescence recovery mechanism can enhance the duration of fluorescence blinking. Our study uses AF647 dye conjugated with Phalloidin antibody in U87MG cell line mounted on Mowiol and β - ME. On the basis of the investigation we optimize the intensity, at the sample plane, of fluorescence excitation laser at 638 nm and fluorescence recovery beam at 405 nm or in the UV giving the maximum possible fluorescence blinking duration. We observe that the longer blinking duration, using the optimized illumination scheme, has brought down the resolution in the super-resolved image, as given by Fourier Ring Correlation method, from 168 nm to 112 nm, while the separation between two nearby resolvable filaments has been brought down to ≤ 60 nm.