TY - JOUR AU - Isotta, Sturniolo AU - Csongor, Váróczy AU - Ákos, Máté Bede AU - Hegedűs, Csaba AU - Demény, Máté Ágoston AU - Virág, László TI - Quantifying antibody-dependent cellular cytotoxicity in a tumor spheroid model : application for drug discovery JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP PY - 2024 SN - 1940-087X UR - https://m2.mtmt.hu/api/publication/34820104 ID - 34820104 LA - English DB - MTMT ER - TY - JOUR AU - Kriel, Y. AU - Kwintowski, A. AU - Feka, K. AU - Windsor, M. AU - Young, G. AU - Walker, M. AU - Askew, C.D. TI - Near-Infrared Spectroscopy During Reactive Hyperemia for the Assessment of Lower Limb Vascular Function JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2024 PY - 2024 IS - 205 SN - 1940-087X DO - 10.3791/66511 UR - https://m2.mtmt.hu/api/publication/34794991 ID - 34794991 N1 - VasoActive Research Group, School of Health, University of the Sunshine Coast, Australia Centre for Trials Research, College of Biomedical and Life Sciences, Cardiff University, United Kingdom Sunshine Coast Health Institute, Sunshine Coast Hospital and Health Service, Australia Export Date: 17 April 2024 Correspondence Address: Kriel, Y.; VasoActive Research Group, Australia; email: ykriel@usc.edu.au AB - Vascular diseases of the lower limb contribute substantially to the global burden of cardiovascular disease and comorbidities such as diabetes. Importantly, microvascular dysfunction can occur prior to, or alongside, macrovascular pathology, and both potentially contribute to patient symptoms and disease burden. Here, we describe a non-invasive approach using near-infrared spectroscopy (NIRS) during reactive hyperemia, which provides a standardized assessment of lower limb vascular (dys)function and a potential method to evaluate the efficacy of therapeutic interventions. Unlike alternative methods, such as contrast-enhanced ultrasound, this approach does not require venous access or sophisticated image analysis, and it is inexpensive and less operator-dependent. This description of the NIRS method includes representative results and standard terminology alongside the discussion of measurement considerations, limitations, and alternative methods. Future application of this work will improve standardization of vascular research design, data collection procedures, and harmonized reporting, thereby enhancing translational research outcomes in the areas of lower limb vascular (dys)function, disease, and treatment. © 2024 JoVE Journal of Visualized Experiments. LA - English DB - MTMT ER - TY - JOUR AU - Cahalane, R.M.E. AU - Turner, M.E. AU - Clift, C.L. AU - Blaser, M.C. AU - Bogut, G. AU - Levy, S. AU - Kasai, T. AU - Driedonks, T.A.P. AU - Nolte-‘T, Hoen E.N.M. AU - Aikawa, M. AU - Singh, S.A. AU - Aikawa, E. TI - Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2024 PY - 2024 IS - 205 SN - 1940-087X DO - 10.3791/66126 UR - https://m2.mtmt.hu/api/publication/34770368 ID - 34770368 N1 - Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, United States Mechanobiology and Medical Device Research Group (MMDRG), Biomedical Engineering, College of Science and Engineering, University of Galway, Ireland Department of CDL Research, University Medical Center Utrecht, Netherlands Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Netherlands Center for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, United States Export Date: 4 April 2024 Correspondence Address: Aikawa, E.; Center for Interdisciplinary Cardiovascular Sciences, United States; email: eaikawa@bwh.harvard.edu LA - English DB - MTMT ER - TY - JOUR AU - Berkane, Y. AU - Jain, R. AU - Ajenu, E.O. AU - Shamlou, A.A. AU - Nguyen, K. AU - McCarthy, M. AU - Uygun, B.E. AU - Lellouch, A.G. AU - Cetrulo, C.L. Jr. AU - Uygun, K. AU - Randolph, M.A. AU - Tessier, S.N. TI - A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2024 PY - 2024 IS - 204 SN - 1940-087X DO - 10.3791/66362 UR - https://m2.mtmt.hu/api/publication/34780018 ID - 34780018 N1 - Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, Harvard Medical School, United States Shriners Children’s Boston, United States Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University, France SITI Laboratory, UMR, INSERM, Université de Rennes, France Center for Engineering in Medicine and Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, United States Innovative Therapies in Haemostasis, INSERM, UMR-S, University of Paris, France Export Date: 9 April 2024 Correspondence Address: Tessier, S.N.; Shriners Children’s BostonUnited States; email: sntessier@mgh.harvard.edu LA - English DB - MTMT ER - TY - JOUR AU - Priedols, M. AU - Paidere, G. AU - Kaukis, P. AU - Bajo-Santos, C. AU - Spule, A. AU - Miscenko, A. AU - Mozolevskis, G. AU - Rimsa, R. AU - Abols, A. TI - Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2024 PY - 2024 IS - 204 SN - 1940-087X DO - 10.3791/66019 UR - https://m2.mtmt.hu/api/publication/34721402 ID - 34721402 N1 - Latvian Biomedical Research and Study Centre, Latvia Institute of Solid-State Physics, University of Latvia, Latvia Cellbox labs LTD, Atari Faculty of Materials Science and Applied Chemistry, Institute of General Chemical Engineering, Riga Technical university, Latvia Export Date: 5 March 2024 Correspondence Address: Bajo-Santos, C.; Latvian Biomedical Research and Study CentreLatvia; email: cristina.bajo@biomed.lu.lv Chemicals/CAS: Polymers Funding details: 739508 Funding details: Latvijas Zinātnes Padome, lzp-2019/1-0142, lzp-2022/1-0373 Funding text 1: We thank all the donors who participated in this study, the staff of the Latvian Genome Database for providing the samples. The Institute of Solid-State Physics, University of Latvia as the Center of Excellence has received funding from the European Union's Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamongPhase2 under grant agreement No. 739508, project CAMART2. This work was supported by The Latvian Council of Science Project No. lzp-2019/1-0142 and Project No: lzp-2022/1-0373. AB - Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements. © 2024 JoVE Journal of Visualized Experiments. LA - English DB - MTMT ER - TY - JOUR AU - McCloskey, Molly C. AU - Kasap, Pelin AU - Trempel, Michelle AU - Widom, Louis P. AU - Kuebel, Julia AU - Chen, Kaihua AU - Gaborski, Thomas R. AU - Engelhardt, Britta AU - McGrath, James L. TI - Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP PY - 2024 IS - 203 SN - 1940-087X DO - 10.3791/65258 UR - https://m2.mtmt.hu/api/publication/34509114 ID - 34509114 LA - English DB - MTMT ER - TY - JOUR AU - Yelleswarapu, M. AU - Spinthaki, S. AU - de, Greef T.F.A. AU - Eduati, F. TI - Bilayer Microfluidic Device for Combinatorial Plug Production JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2023 PY - 2023 IS - 202 SN - 1940-087X DO - 10.3791/66154 UR - https://m2.mtmt.hu/api/publication/34632373 ID - 34632373 N1 - Export Date: 19 February 2024 Correspondence Address: Eduati, F.; Department of Biomedical Engineering, Netherlands; email: f.eduati@tue.nl Funding details: Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO, 24.005.009 Funding text 1: We would like to thank Stacey Martina of the NanoLab TuE for help with HMDS vapour deposition. This research was funded by the Institute for Complex Molecular Systems (ICMS) at TU/ e and by the Netherlands Organization for Scientific Research (NWO) Gravitation programme IMAGINE! (project number 24.005.009). AB - Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions-from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies. © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. LA - English DB - MTMT ER - TY - JOUR AU - Jayaswal, Praapti AU - Ilyas, Mohd AU - Singh, Kuljit AU - Kumar, Saurabh AU - Sisodiya, Lovely AU - Jain, Sapna AU - Mahlawat, Rahul AU - Sharma, Nishant AU - Gupta, Vishal AU - Atmakuri, Krishnamohan TI - Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP PY - 2023 IS - 202 PG - 22 SN - 1940-087X DO - 10.3791/65138 UR - https://m2.mtmt.hu/api/publication/34613123 ID - 34613123 AB - Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm. LA - English DB - MTMT ER - TY - JOUR AU - Wagner, Jackson C. AU - Yang, Bin AU - Wu, Zishan AU - Xiong, Wei TI - Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP PY - 2023 IS - 202 PG - 18 SN - 1940-087X DO - 10.3791/65388 UR - https://m2.mtmt.hu/api/publication/34611670 ID - 34611670 AB - Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of -100 mu m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to -1 mu m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of noncentrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemicalspecific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging. LA - English DB - MTMT ER - TY - JOUR AU - Carver, J.J. AU - Didonna, A. TI - Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis JF - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS J2 - JOVE-J VIS EXP VL - 2023 PY - 2023 IS - 202 SN - 1940-087X DO - 10.3791/66218 UR - https://m2.mtmt.hu/api/publication/34564284 ID - 34564284 N1 - Export Date: 7 February 2024 Correspondence Address: Didonna, A.; Department of Anatomy and Cell Biology, United States; email: didonnaal21@ecu.edu Chemicals/CAS: Autoantibodies; Cytokines Funding details: National Institutes of Health, NIH, R03NS131908 Funding details: U.S. Department of Defense, DOD, W81XWH-22-1-0517 Funding details: East Carolina University, ECU Funding text 1: This study was supported by the National Institutes of Health (R03NS131908) and the Department of Defense through the Multiple Sclerosis Research Program under Award No. W81XWH-22-1-0517. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This study was also supported by East Carolina University startup funds. AB - Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination. © 2023 JoVE Journal of Visualized Experiments. LA - English DB - MTMT ER -