Immunodiagnostics

This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

This video showcases a flow cytometric gating strategy utilizing a multi-color panel to analyze cell viability and specific fluorescent-conjugated anti-human antibody markers. This approach makes it possible to determine the percentage of Mycobacterium tuberculosis-infected cell populations accurately.

This video demonstrates the staining and preparation of bacterial cells for immunological studies using red fluorescent dye. The stained bacteria are diluted and resuspended in a growth medium to achieve the desired bacterial concentration for host cell infection, representing the multiplicity of infection.

This video demonstrates the detection of vitronectin's interaction with bacterial surface proteins. The flow-cytometry analysis measures the fluorescence signal of fluorescently labeled antibodies bound to vitronectin, confirming its presence at the bacterial surface. This technique provides insights into host-pathogen interactions and potential bacterial infection therapies.

This video demonstrates the evaluation of stromal cytokine-induced intracellular phosphorylation of proteins in leukemia cells. These intracellular phosphorylated proteins are quantified using the phospho-flow cytometry technique.

In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

This video demonstrates a technique for nucleic acid extraction from whole human blood using magnetic glass particles (MGPs) — small magnetic beads coated with silica. Upon lysing the blood cells using a lysis buffer, the released nucleic acids bind to the surface of silica-coated MGPs, extracting the molecules from the sample.

This video demonstrates mast cell exocytosis induced by an activating reagent that includes a calcium ionophore, initiating the release of secretory granules containing the fluorescent reporter protein NPY-mRFP. Subsequently, we quantitatively assess the exocytosis by measuring the released NPY-mRFP using a fluorescence plate reader.

This video demonstrates the method to analyze energy metabolism in activated human NK cells using an extracellular flux analyzer. Different inhibitors of the electron transport chain complexes impact ATP synthesis, which is measured through the oxygen consumption rate or OCR, providing insights into cellular metabolism and bioenergetics.

This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

This video demonstrates the detection of the Rhesus (Rh) factor in blood samples using a magnetic levitation instrument. The magnetic field aligns and levitates beads and blood cells based on density. Treating the samples with anti-Rh antibodies results in intermediate-density bead-cell complexes' formation, allowing for the differentiation of Rh-positive and Rh-negative red blood cells.

This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

This video demonstrates a method of decaging a photoactivatable peptide-major histocompatibility complex using UV irradiation. The decaging process exposes the native peptide sequence, enabling precise T cell activation.

This video demonstrates a technique for the detection of extrapulmonary tuberculosis. Superparamagnetic iron oxide (SPIO) nanoprobes, conjugated to Mycobacterium-specific antibodies, are injected into a mouse previously infected with Mycobacterium bovis BCG, labeling the granuloma and providing a better imaging contrast for its detection.

This video demonstrates a method to study the uptake and retrograde transport of functionalized nanobodies to understand their intracellular trafficking pathways. By introducing radiolabelled sulfate and utilizing isolation and detection techniques, this method enables the study of endocytic uptake and trans-Golgi network arrival of cargo proteins.

This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic chip.

This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.

This video illustrates a bead-based multiplex assay for analyzing cytokines in human tears. The method allows for the simultaneous analysis of multiple cytokines from a single sample, providing enhanced sensitivity for studying immune responses in various pathophysiological conditions.

This video demonstrates a method for assessing alloreactivity in mixed T cell populations by observing interactions with alloantigen-carrying dendritic cells, leading to the formation of immunological synapses. Differential staining and quantification of synapse-containing doublets via flow cytometry reveal T cell alloreactivity.

This video demonstrates the use of flow cytometry to identify and count various immune cells in bronchoalveolar lavage fluid. Initially, single immune cells are identified using a viability dye, followed by differential staining of surface marker proteins, enabling the precise enumeration of each cell type.

This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these cells.

This video demonstrates a laminar flow-based assay to characterize the interaction of leukocytes and cellular partners such as endothelial cells. This method provides dynamic insights into adhesion, deadhesion, and transmigration of leukocytes when perfused over an intact and confluent layer of vascular cells.

The video demonstrates an assay for assessing bulk autophagic sequestration activity in mammalian cells. It utilizes electroporation to release cytosolic proteins, including lactate dehydrogenase (LDH). The sequestered LDH and total cellular LDH fractions are determined to assess LDH sequestration, providing insights into the overall bulk sequestration activity.

This video demonstrates a reporter cell-based assay for screening potential bioactive nanoparticles that inhibit Toll-like receptor (TLR) signaling. The assay involves introducing lipopolysaccharides (LPS) mixed with peptide-gold nanoparticle hybrids to reporter macrophages expressing the reporter proteins — secreted embryonic alkaline phosphatase (SEAP) and luciferase. The reduction of LPS-induced TLR signal mediated by the hybrids is determined by measuring the reporter signals.

This video demonstrates the method to assess the impact of shear stress on platelets. Antibody treatment induces dissociation and conformational changes in mechanoreceptor complexes, activating the platelets and expressing markers on the surface. These markers are observed via immunostaining and flow cytometry.

This video demonstrates the measurement of trans-epithelial electrical resistance in irradiated Caco-2 cells, both in the presence and absence of PBMC cells. This method enables the study of the synergistic effects of radiation and immune cells on the epithelial cell barrier.

This video demonstrates an assay to study the plasma membrane resealing efficiency in mammalian cells. The cells are exposed to a bacterial pore-forming toxin, which forms pores in the cell membrane. In the presence of extracellular calcium ions, the pore causes calcium influx, inducing membrane-resealing events. The resealing efficiency is assessed using DNA-binding propidium iodide dye, which only enters cells without a resealed membrane.

This video demonstrates immunohistochemistry staining for rabies virus antigen detection in infected mouse brain tissue. The process includes deparaffinization and alcohol treatment, followed by proteolytic treatment to enhance antigen accessibility. Immunohistochemistry staining reveals red precipitates, confirming the presence of rabies antigens in the tissue section.

This video demonstrates a technique to quantify the relative ratio of macrophages among the total infiltrated immune cell populations in temporal artery biopsy. The immunohistochemical staining of a tissue section affected by autoimmune disease selectively stains the immune cells with specific receptors to visually differentiate them from the rest of the cells.

This video demonstrates the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying RNA-binding proteins in mouse testis. eCLIP enables the precise identification of binding sites where RNA-binding proteins (RBPs) interact with their target RNAs.

This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and a nucleic acid stain.

This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, γδ T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin,...

This video illustrates a cellular assay involving glioblastoma cells expressing CXCR4 chemokine receptors. The assay employs a calcium-sensitive dye to measure fluorescence changes upon agent interaction, offering insights into cellular signaling dynamics.

This video demonstrates an assay investigating the role of neutrophil-like cells in safeguarding lymphoma cells against the cytotoxicity of chemotherapeutic agents. A 3D co-culture, consisting of lymphoma and neutrophil-like cells, is established and exposed to vincristine—a chemotherapy drug. Direct contact with the neutrophil-like cells induces an upregulation of anti-apoptotic proteins in the lymphoma cells, thereby promoting their survival in the presence of vincristine.

The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

The video showcases a flow cytometry-based assay for assaying immunodominance in cytotoxic T lymphocytes or CTL responses. Injected fluorescent target cells exhibit varying fluorescence corresponding to immunogenic epitopes. Post-spleen collection and sorting reveal a reduction in high-intensity fluorescent cells, confirming immunodominance in CTL responses.

The video demonstrates the electrophysiological analysis of embryonic stem cell-derived neurons after exposure to neurotoxins. Treating these neurons with botulinum neurotoxins reduces the frequency of spontaneous miniature excitatory post-synaptic currents, confirming inhibition of synaptic transmission.

Source:Uju L. Madu1, Maphori Maliehe1, Nozethu Mjokane1, Olihile M. Sebolai11Department of Microbiology and Biochemistry, University of the Free State.The video demonstrates evaluating photodynamic treatment's impact on murine macrophage phagocytic efficiency with primaquine. Treated, infected macrophages generate reactive oxygen species, leading to selective fungal death, evident by reduced colonies on the plate.

Source:Uju L. Madu1, Nozethu Mjokane1, Maphori Maliehe1, Olihile M. Sebolai11Department of Microbiology and Biochemistry, University of the Free State.The video demonstrates a technique for evaluating the influence of photodynamic treatment (PDT) with a photosensitizer on the metabolic activity of murine macrophages. The cells undergo treatment with the photosensitizer followed by exposure to UV light, initiating PDT. This process triggers the photosensitizer to generate reactive oxygen species...

Source:Adepemi O. Ogundeji1, Nozethu Mjokane1, Maphori Maliehe1, Olihile M. Sebolai11Department of Microbiology and Biochemistry, University of the Free State.This video showcases a method to assess the impact of aspirin and ibuprofen, at their minimum inhibitory concentrations, on macrophage phagocytosis using pH-sensitive fluorescent dye-stained fungal cells. The drugs inhibit cyclooxygenase-2, decreasing prostaglandin formation and boosting macrophage phagocytosis, as observed through...

Source:Tia Rizakos1, Jennifer Geddes-McAlister11Molecular and Cellular Biology Department, University of Guelph.This video demonstrates the protein isolation from neutrophils, which is further processed and digested into smaller peptides. These peptides can be used for future analysis.

Source:Tia Rizakos1, Jennifer Geddes-McAlister11Molecular and Cellular Biology Department, University of Guelph.This video demonstrates peptide purification through the STAGE Tip procedure. A miniature column containing resin beads bonded to long hydrocarbon chains facilitates hydrophobic interaction-based purification. Upon loading a pre-digested peptide sample into the column, the peptide fragments bind to the resin beads, allowing impurities to pass through. An elution buffer that disrupts...

This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.

This video demonstrates in vitro stimulation of macrophage extracellular traps (METs) in human monocyte-derive macrophages using different inflammatory stimuli such as TNF-alpha, PMA, and HOCL. The video also demonstrates the method of SYTOX dye staining of METs for visualization.

This video demonstrates the reporter enzyme fluorescence (REF) imaging for in vivo detection of Mycobacterium tuberculosis infection. The infected mice are intraperitoneally injected with a substrate for REF imaging. Upon being hydrolyzed by bacterial β-lactamase enzyme, the substrate emits fluorescence, the detection of which aids evaluation of infection.

This video investigates Leishmania's virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania's virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

This video demonstrates a method for assessing the intracellular growth of GFP-tagged Coxiella mutants within cultured epithelial cells. The mutant phenotype is visualized using immunostaining and epifluorescence microscopy, allowing the study of the effect of the mutation on intracellular growth.

In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.

This video demonstrates an in vivo imaging technique for detecting and quantifying eye inflammation in a mouse model of post-infectious uveitis. The technique, known as optical coherence tomography (OCT), generates high-resolution cross-sectional images of the eye for assessing disease progression.

This video demonstrates the visualization of bacteria in deparaffinized bladder tissue from a urinary tract-infected patient. Fluorescently-labeled probe hybridized with bacterial 16s ribosomal RNAs reveals the tissue-associated bacteria and their distribution, providing valuable spatial insights into their density within the sample.

This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes' migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

This video demonstrates label-free identification of lymphocyte morphology and biochemistry through 3D quantitative phase imaging. This technique allows for a thorough analysis of lymphocytes, uncovering their internal structures and properties without the need for labeling procedures.

This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran — a fluorescent reporter — which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium's higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.

The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

The video demonstrates the use of photoconversion to study inflammatory cell dynamics in Drosophila pupae. Green hemocytes are drawn to the wounded epithelium, followed by distant migration. Photoconversion shifts certain hemocytes from green to red, facilitating their movement tracking.

This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

This video demonstrates an in vivo immunofluorescence localization (IVIL) technique to study the biodistribution of tumor-targeting antibodies in a murine model of breast cancer. Upon the intravenous injection of a tumor-specific antibody in the mouse, the tumor is surgically isolated, and the localization of the antibody within the tumor tissue is confirmed via ex vivo immunostaining.

This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.

This video demonstrates localized surface plasmon resonance imaging to detect protein secretions from a single cell. Using a microfluidic setup and a peptide-bound plasmonic nanostructure chip, changes in reflected light intensity at the resonance angle reveal interactions with secreted proteins from a single cell.

This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

This video demonstrates an in vivo assay designed to measure bacterial phagocytosis in adult Drosophila flies. Following the injection of fluorescently labeled bacteria into the abdomen of adult flies, hemocytes associated with the dorsal vessel engage in phagocytosis. The introduction of trypan blue dye serves to quench the fluorescence emitted by extracellular, non-phagocytosed bacteria. This quenching process enhances the identification of phagocytosed bacteria when observed under a...

The video demonstrates a method for assessing receptor forces applied by immune cells using polyethylene glycol-anchored gold nanoparticles and DNA hairpin tension probes. CD8-positive T cell interactions trigger probe unfolding, emitting fluorescence while locking strands sustain the signal for quantifying the forces applied by immune cells.

This video demonstrates a colorimetric E. coli detection method, involving an E. coli biomarker activating a DNAzyme, leading to cleavage of RNA linkage and detachment of a DNAzyme-urease conjugate. A subsequent colorimetric assay detects the E. coli biomarker through ammonia-induced pH changes.

This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5' Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader...

This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

This video describes the imaging of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Source:Matthew J Murray1, Gary R McLean2,3, Matthew B Reeves11Institute of Immunity and Transplantation, University College London.2Cellular and Molecular Immunology Research Centre, London Metropolitan University.3National Heart and Lung Institute, Imperial College London.This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The...

Source:Lale Ertuglu1, Michael Pridmore2, Rachelle Crescenzi2,3, Annet Kirabo41Division of Nephrology and Hypertension, Vanderbilt University Medical Center.2Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center.3Department of Biomedical Engineering, Vanderbilt University.4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center.This video demonstrates the quantification of sodium content in human tissues using sodium...

This video demonstrates a method to establish a gastric organoid infection model by microinjecting gastric pathogen Helicobacter pylori into the lumen of the organoids. This model helps in studying infection-induced changes in gastric cells.

This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal's back region for biofilm development.

This video demonstrates the development of necrotizing enterocolitis disease in a human-derived neonatal enteroid model induced by lipopolysaccharides treatment. This model helps to study intestinal pathophysiology.

In this video, we demonstrate a tail-snipping procedure in a mouse model to collect peripheral blood. The collected blood is subsequently subjected to centrifugation to separate plasma, which can be used for further experiments.

This video demonstrates a method to induce apoptosis in the Jurkat cells — a human T lymphocyte leukemia cell line. The cells are exposed to ultraviolet C (UVC) radiation, which causes extensive DNA damage and leads to apoptotic cell death.

In this video, we describe a procedure to inflate isolated human lungs with agarose and prepare human precision-cut lung slices. The prepared ex vivo lung slices can be used for further immunologic experiments.

This video illustrates a technique for evaluating cytotoxicity in human precision-cut lung slices through the WST-1 assay. Human precision-cut lung slices are subjected to incremental concentrations of a cytotoxic chemical to quantify their cytotoxicity. A decrease in color intensity serves as confirmation of the chemical's detrimental impact on lung cells.

This video demonstrates the isolation and concentration of bacterial culture filtrate proteins, which include bacterial extracellular vesicles, using ultrafiltration. The concentrated extracellular vesicles serve as diagnostic markers for disease detection.

This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

This video demonstrates the seedling growth inhibition assay in the Arabidopsis thaliana plant. The impact of increased concentration of immune peptide elicitor on seedling growth is determined.

This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

The video demonstrates a procedure for quantifying the Zika virus in multiple mouse organs via a focus-forming assay. The process involves infecting mammalian cell monolayers with virus-infected tissue homogenates, followed by the use of methyl cellulose to prevent viral spread. Subsequently, immunostaining is employed to enable precise quantification and measurement of organ-specific infectivity.

This video demonstrates the detection of bacteriocin-producing lactic acid bacteria derived from mouse fecal matter. The assay determines the antimicrobial activity of bacteriocin on specific bacterial strains.

This video demonstrates the riboflavin and UV light-based pathogen reduction process against high-titer bacterial contamination in human platelet products. This process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

This video demonstrates the in vitro measurement of enzyme activity for α-galactosidase A and acid α-glucosidase in samples using synthetic 4-Methylumbelliferyl substrates.

This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

The video demonstrates a method for investigating the impact of stromal cells on leukocyte recruitment. In a parallel plate flow chamber model, pro-inflammatory cytokines induce the expression of adhesion receptors by endothelial cells, facilitating neutrophil recruitment and migration. The cross-talk between stromal cells and endothelial cells diminishes neutrophil recruitment to the endothelial cells.

This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

This video demonstrates a method to study natural killer (NK) cell migration through a porous membrane insert system in response to chemoattractants. This method enables quantitative analysis of NK cell migration using flow cytometry.

This video demonstrates a technique to evaluate the complement-mediated bactericidal activity of antibodies present in serum. This approach combines isolated serum with pathogenic bacteria and exogenous complement proteins. Upon incubation, the bacteria are grown on an agar plate, and the produced colonies are quantified to measure the serum bactericidal titer.

The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

The video shows an in vitro opsonophagocytic killing assay using a co-culture of human neutrophils with Streptococcus pneumoniae. Antibodies and complement proteins facilitate opsonization of the bacteria, enabling neutrophils to recognize and kill bacteria, showing successful neutrophil opsonophagocytic activity.

In this video, we demonstrate an assay to assess the survival of Porphyromonas gingivalis inside human endothelial cells. The measure of survival and growth of internalized Porphyromonas gingivalis is evaluated by the number of colonies on the blood agar plate.

This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

In this video, we demonstrate an assay utilizing fluorescence microscopy to detect the adhesion of cancer cells to neutrophil extracellular traps (NETs). In the test wells, there is a notable increase in the number of fluorescently labeled cancer cells adhering to NETs, whereas wells treated with DNase exhibit reduced cancer cell adhesion to NETs.

This video demonstrates the lactate dehydrogenase, LDH assay to measure the cytotoxic effect of a test substance and its effect on the membrane integrity in human precision-cut lung slices. The assay provides a quantitative measure of cell death or damage in the sample.

This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds' stimulatory and inhibitory effects, respectively.

This video showcases the assessment of B cell activation through the utilization of antigenic liposomes. B cells treated with calcium-sensitive Indo-1 dye are distinguished using flow cytometry, and their violet-to-blue fluorescence ratio is analyzed. As the cells encounter antigenic liposomes, it triggers B cell activation through calcium influx, progressively enhancing the violet-to-blue fluorescence signal ratio over time.

This video demonstrates a method to quantify fungal infection in zebrafish larvae. Fungal spores are microinjected into the larval hindbrain ventricle, and the larvae are incubated to allow infection development. Infected larvae are harvested at defined intervals, and infection progress is assessed by enumerating colony-forming units.

This video demonstrates an opsono-adherence assay for examining the interaction between opsonized bacteria and immune cells. Fluorescently labeled bacteria, treated with test serum containing anti-capsule antibodies, are incubated with macrophages. After staining the macrophages with fluorescence, the attached bacteria on the macrophage surface are observed under a microscope for visualization.

This video demonstrates a bead-based multiplex immunoassay technique to simultaneously detect antibodies for multiple environmental pathogens in human saliva. Fluorescent beads were coupled to antigens from different pathogens, which were captured using antibodies in the human saliva sample. Upon labeling the antibodies using a fluorescent reporter, the beads were analyzed using flow cytometry to assess the presence of different pathogen-specific antibodies in the saliva.

This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

This video demonstrates the quantification of immune mediator levels in neonatal human nasal mucosal lining fluid extract. An electrochemiluminescence-based multiplex assay system measures these mediators by evaluating the intensity of emitted light when they are excited.

This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

This video demonstrates an in vitro assay to quantify neutrophil serine protease activity in sputum samples. The sample containing secreted protease is incubated with a fluorescent reporter bearing a recognition motif. Cleavage of the motif by the protease enables individual fluorescence emission by the donor and acceptor fluorophore of the reporter, indicating protease activity in the sample.

This video showcases an apoptosis assay in cancer cells induced by pre-activated effector T cells. By co-culturing these cells with cancer cells marked with fluorescent nuclei, a caspase-cleavable fluorogenic substrate is added. Apoptotic cancer cells will display a different color in their nuclei, leading to dual fluorescence. This dual fluorescence is indicative of cancer cells undergoing apoptosis.

This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing α2,6-linked sialic acid in human sera. This assay is often used for influenza human...

This video demonstrates an assay to measure oxidative burst in Arabidopsis leaves upon immune elicitation. Leaf discs, cut from plant leaves, are incubated with bacterial peptides functioning as microbe-associated molecular patterns or MAMPs. Upon detection of the MAMPs, plant cells induce the production of extracellular ROS, which is detected using a chemiluminescence assay.

This video demonstrates an assay for real-time detection of reactive oxygen species (ROS) production in rice tissues upon pathogen-associated molecular pattern (PAMP) elicitation. Cut sheath segments and leaf discs are exposed to a bacterial flagella-derived peptide functioning as a PAMP. Upon recognition of the peptide via pattern recognition receptors (PRRs) on plant cells, a rapid and transient production of ROS occurs, which is detected via a chemiluminescence assay.

This video showcases a monocyte monolayer assay utilizing primary monocytes from peripheral blood to assess Fcγ receptor-mediated phagocytosis with poly D antibody-opsonized R2R2 red blood cells.

This video showcases the Charcoal Agar Resazurin Assay (CARA) for evaluating the activity of antimicrobial compounds against Mycobacterium tuberculosis. CARA aids in assessing the efficacy of these compounds by gauging their impact on bacterial growth and distinguishing between bactericidal and bacteriostatic effects.

This video demonstrates an assay to examine the toll-like receptor(TLR)-dependent activity of the transcription factors nuclear factor-kappa-B (NF-κB) and activator protein-1 (AP-1) in macrophages. The TLRs trigger a pro-inflammatory signaling cascade upon recognizing damage-associated molecular patterns (DAMPs), activating NF-κB and AP-1. The engineered reporter macrophages produce secreted embryonic alkaline phosphatase (SEAP) enzyme upon NF-κB and AP-1 activation, which is detected via a...

This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic capabilities.

This video demonstrates a technique for assessing the efficacy of antibodies against tumor necrosis factor-alpha (TNFα), an inflammatory cytokine, through an in vitro bioanalytical approach. TNFα, when introduced to stressed cells, triggers apoptosis. However, binding to the antibodies confines TNFα, averting apoptosis. The degree of apoptosis is quantified using a bioluminescence assay.

This video demonstrates a monocyte-derived dendritic cell, MoDC-based assay to measure the immunogenicity of commercially available vaccines. MoDC assays investigate dendritic cell responses to antigens, pathogens, or treatments and can provide insights into immune responses, vaccine development, and immunotherapy research.

This video demonstrates the hemocyte disturbance assay, evaluating hemocyte re-adhesion in Drosophila larvae's hematopoietic pockets. Mechanical disturbance releases resident hemocytes, elevating circulating hemocyte counts. Post-recovery, re-adherence of resident hemocytes is confirmed, illustrating reversibility.

This video demonstrates an assay to measure antibody-dependent enhancement (ADE) of Dengue virus infection using Zika Virus Convalescent Serum. The assay uses Dengue reporter viral particles (RVPs) encoding a green fluorescent protein. Serum antibodies against the Zika Virus cross-react with RVPs to form a complex. The bound antibodies enhance RVP uptake by cells expressing the Fc receptor, which is detected using flow cytometry.

This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the...

This video demonstrates the dye release assay for quantifying the enzymatic activity of antimicrobial proteins. The antimicrobial protein degrades the bacteria's dye-labeled cell wall and releases the dye molecules turning the solution blue. The absorbance of the solution correlates with the enzymatic activity of the antimicrobial protein.

This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing strains.

This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm's metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells.

This video demonstrates an assay for monitoring dendritic cell migration following footpad infection with Mycobacterium bovis Bacille Calmette-Guérin BCG in mice. Upon bacterial infection, activated skin DC cells are labeled with a cell-permeable carboxyfluorescein diacetate succinimidyl ester (CFSE) dye. After a suitable interval for DC migration to the popliteal draining lymph node, the node cells are isolated and subjected to immunostaining for DC-specific surface markers. Identification of...

This video illustrates the reconstitution of synthetic antigen-presenting cells through Bead-Supported Lipid Bilayers and their application in assessing the synaptic output generated by activated T cells.

This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

This video demonstrates turbidimetric analysis to assess collagen-induced platelet aggregation in pre-treated, washed human platelets. Control wells show collagen-induced aggregation, whereas a high concentration of inhibitors blocks Pannexin-1 channels, thereby inhibiting collagen-induced platelet aggregation, evident from unchanged turbidity.

This video demonstrates the viral inactivation assay, showcasing the evaluation of the impact of antiviral agents on viral particles.

This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows...

The video demonstrates an assay for quantifying influenza viruses A and B. This assay employs nicking endonuclease amplification with influenza-specific primers and target detection using molecular beacon probes.

This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor of the production of pyomelanin by the bacteria, the optical density of the wells is measured to compute the minimum inhibitory concentration of the antibiotic, and the impact of the...

This video demonstrates an assay to study the effect of extracellular matrix (ECM) stiffness on bacterial infection of adherent cells. Polyacrylamide hydrogels of varying stiffness, overlaid with collagen, are layered in a multi-well plate to mimic the stiffness of physiological ECM. Upon overlaying human endothelial cells on the hydrogels and infecting the cells with Listeria monocytogenes bacteria engineered to express a fluorescent protein once intracellular, flow cytometry is used to...

This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.

This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.

This video describes the simple ATP-measuring assay using luciferase glow reagent. This assay is used to quantify Neisseria gonorrhoeae survival after treatment with ceftriaxone.

This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a β-galactosidase substrate. The substrate is cleaved by active lysosomal β-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

This video demonstrates a technique to detect bacterial contaminants in food and water samples using nanosensors. The target pathogenic bacteria are detected through a combination of magnetic relaxation and fluorescence emission modalities.

The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

This video demonstrates a fluorescence-based method for quantifying bile salt-induced biofilm formation. Upon the addition of bile salts in the culture of a pathogenic enteric bacteria, biofilm production is detected using a fluorescent lectin that binds to the polysaccharides in the extracellular polymeric substance (EPS) matrix of the biofilm.

The video showcases a biofilm dispersion assay using a multi-well plate. Buffer and glucose treatment disassemble the biofilm, releasing bacteria, while bile salts induce stress, retaining bacteria in the protective extracellular polymeric substance. Higher dispersion with buffer and glucose confirms successful biofilm disassembly, estimated by colony forming units measurement.

This video demonstrates a real-time assay for monitoring extracellular pH in dental biofilms using a pH-sensitive ratiometric probe. When an in situ-grown bacterial biofilm is exposed to glucose, a reduction in extracellular pH near the bacterial biomass protonates the probe, leading to its increased concentration inside the bacteria. The disparity in emission wavelength between the protonated and unprotonated probe is utilized to calculate the pH across the extracellular matrix of the biofilm...

The video demonstrates a fluorescence resonance energy transfer (FRET) flow cytometry assay to detect the seeding activity of protein aggregates isolated from biological samples. Mammalian cells expressing tau reporter proteins are incubated with liposome transduction complexes containing tau seeds. These seeds mediate the aggregation of the reporter proteins, leading to generating a FRET positive signal in the flow cytometer.

Source:Rhea Kang1, Gabby Mora1, Sherry Dunbar11Luminex, A DiaSorin Company, Austin.This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.

Source:Kelly Da Costa1, Diego Cantoni1,2, Martin Mayora-Neto1, Tobia Fantoni1,3, Nigel Temperton11Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent.2MRC-University of Glasgow Centre for Virus Research, University of Glasgow.3Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona.This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and...

Source:Jing Li1, Jaehyung Cho2,31MBL International Corporation, Des Plaines.2Division of Hematology, Department of Medicine, Washington University School of Medicine.3Department of Pathology and Immunology, Washington University School of Medicine.This video demonstrates an assay to monitor neutrophil activation and granule exocytosis through a degranulation assay. Upon activation by proinflammatory compounds, cytoplasmic granules within neutrophils containing gelatinase fuse with the cell...

Source:Jing Li1, Jaehyung Cho2,31MBL International Corporation, Des Plaines.2Division of Hematology, Department of Medicine, Washington University School of Medicine.3Department of Pathology and Immunology, Washington University School of Medicine.This video demonstrates a degranulation assay of azurophilic granules in mouse neutrophils, involving the stimulation of neutrophils to release myeloperoxidase-containing azurophilic granules. The subsequent enzymatic reaction is employed to quantify...

Source:Tumininu S. Faniyan1, Dennis J. Samuel1, Kavaljit Chhabra11University of Rochester Medical Center.This video demonstrates a procedure for collecting mouse urine and assessing its glucose levels. To evaluate the glucose level, immerse a test strip coated with enzymes and a chromogenic dye into the sample, initiating a color change that indicates the glucose levels. For quantifying urine glucose levels, introduce a urine sample to a multiwell plate with escalating glucose concentrations,...