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Assessment of fetal genetic disorders
Published in Hung N. Winn, Frank A. Chervenak, Roberto Romero, Clinical Maternal-Fetal Medicine Online, 2021
Teresa Martino, J. Pratt Rossiter, Karin J. Blakemore
In order to identify fetal cells, monoclonal antibodies against various fetal cell antigens have been developed. These include antibodies to trophoblasts (101,102), fetal erythrocyte cell surface antigens (103–105), and paternally derived human leukocyte antigens (106,107). To enrich for fetal cells, the monoclonal antibodies have been fluorescently tagged and used in combination with flow cytometry, a method to sort individual cells based on their specific physical and chemical properties. Characteristics selected for include cell size, cell granularity, and the presence of a fluorescent tag (104,105). By report, multiparameter flow cytometry applied to first-trimester maternal peripheral blood enriches from one fetal nucleated erythrocyte in approximately 107 to 108 maternal cells to approximately 4 fetal cells per 1000 maternal cells (104). The proportion of fetal cells after sorting second-trimester samples was approximately 20 fetal cells per 1000 maternal cells in this study, though other investigators have not described such high yields of fetal cells (108,109). In addition to fluorescence-activated flow cytometry, isolation methods under investigation include immunospecific magnetic-activated cell sorting (110), immunomagnetic beads (111), avidin–biotin columns (112), and discontinuous density-gradient centrifugation (113). The various methods were reviewed and their advantages and disadvantages were analyzed by Holzgreve and colleagues (114).
Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
DNA fragments incorporating the coding sequences of the genes of interest can be cloned in frame with N- or C-terminally tagged GFP and fRFP using established (e.g. pEGFP-N1, Clontech; pmKate2-N, Evrogen) or bespoke vectors. It is recommended that for each target protein, a GFP and an fRFP tagged construct be created because, in our experience, the nature of the protein-tag can have an effect on protein expression, even if the reasons for this are not necessarily clear. It is further important to keep in mind that GFP has severely diminished fluorescence intensity when in acidic compartments (Haupts et al., 1998), such as the lysosome, and that FRET will work poorly or not at all when fluorescent proteins are on opposite sides of the membrane. Therefore, it is recommended to use known protein structures or topologies to place the fluorescent tag on the protein terminus that is cytosolic for each target protein. In cases where protein topology is unknown, both the N- and C-termini should be separately tagged for each fluorescent protein. fRFP is recommended over shorter wavelength RFPs (e.g. DsRed or mCherry, Clontech) because the longer wavelength excitation spectrum of fRFP has essentially no overlap with the 488-nm laser, meaning very little background signal will arise from direct laser stimulation.
Tight junctions: from molecules to gastrointestinal diseases
Published in Tissue Barriers, 2023
Aekkacha Moonwiriyakit, Nutthapoom Pathomthongtaweechai, Peter R. Steinhagen, Papasara Chantawichitwong, Wilasinee Satianrapapong, Pawin Pongkorpsakol
The tight junction (TJ) was first visualized by transmission electron microscopy (TEM) in regions between adjacent epithelial cells in 19631 and was shown to be related to paracellular transport.2–4 Freeze-fracture electron microscopy demonstrated an anastomosing network of TJ strands that were superficial and deep in “leaky” and “tight” epithelia,5,6 respectively. The morphology of TJ strands was found to be strongly correlated with epithelial barrier function, as indicated by the transepithelial electrical resistance (TER) in the intestinal epithelial cells.7 The lipid micelle model was proposed as the first description of the molecular identity of TJs, in which the exoplasmic leaflets of neighboring cell membranes are partly fused together as lipidic hexagonal cylinders,8–10 establishing a membrane fence. Later, it was reported that fluorescent-tag lipids were unable to diffuse in paracellular space,11 thus refuting this hypothesis.
Nanovaccine administration route is critical to obtain pertinent iNKt cell help for robust anti-tumor T and B cell responses
Published in OncoImmunology, 2020
Yusuf Dölen, Michael Valente, Oya Tagit, Eliezer Jäger, Eric A. W. Van Dinther, N. Koen van Riessen, Martin Hruby, Uzi Gileadi, Vincenzo Cerundolo, Carl G. Figdor
PLGA (Resomer RG 502 H, lactide/glycolide molar ratio 48:52 to 52:48) was purchased from Boehringer Ingelheim. Solvents for PLGA preparation (dichloromethane) were obtained from Merck. CryoSure-DMSO from WAK-Chemie. Polyvinyl alcohol (PVA), isopropyl alcohol (IPA, ≥ 99.7%), water for HPLC (H2O), acetonitrile for HPLC (ACN, ≥ 99.9%), methanol for HPLC (MeOH, ≥ 99.9%) and anhydrous chloroform (CHCl3, ≥99%) were obtained from Sigma-Aldrich. Endotoxin-free ovalbumin (OVA) from Hyglos. OVA (257–264) SIINFEKL and HPV16 E7(49–57) were obtained from Anaspec. IMM-60 was kindly gifted by Ian Walters at IOX Therapeutics. Vivotag-S 750 fluorescent tag was purchased from Perkin Elmer and RPMI 1640 medium from Life Technologies Inc. CD3 (145-2C11) was obtained from BD, CD45.1 (A20), CD8α (53–6.7), XCR-1 (ZET), NK1.1 (PK136), CD11 c (N418), CD11b (M1/70), CD40 (3/23), I-A/I-E (M5/114.15.2), CD69 (H1.2F3), CD194-CCR4 (2G12), PD-1 (29 F.1A12) and CD90.1-Thy1.1 (OX-7), CD107a (1D4B), KLRG1 (2F1/KLRG1) antibodies were obtained from BioLegend. eBioscience™ Fixable Viability Dye eFluor™ 780 was purchased from Thermo Fisher. H2-Kb/SIINFEKL and CD1d- α-GalCer dextramers were purchased from Immudex. Celltrace CFSE, Celltrace- violet and Celltrace red were obtained from Invitrogen. For in vivo treatment, anti-PD-1 (RMP1-14), anti-PD-L1 (10 F.9G2), and anti-4-1BB (3H3) was obtained from BioXcell. HPV-16 E7 peptides RAHYNIVTFCCKCDS (LP) and RAHYNIVTF (SP) were obtained from Genscript.
Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop
Published in Journal of Extracellular Vesicles, 2019
Ashley E. Russell, Alexandra Sneider, Kenneth W. Witwer, Paolo Bergese, Suvendra N. Bhattacharyya, Alexander Cocks, Emanuele Cocucci, Uta Erdbrügger, Juan M. Falcon-Perez, David W. Freeman, Thomas M. Gallagher, Shuaishuai Hu, Yiyao Huang, Steven M. Jay, Shin-ichi Kano, Gregory Lavieu, Aleksandra Leszczynska, Alicia M. Llorente, Quan Lu, Vasiliki Mahairaki, Dillon C. Muth, Nicole Noren Hooten, Matias Ostrowski, Ilaria Prada, Susmita Sahoo, Tine Hiorth Schøyen, Lifu Sheng, Deanna Tesch, Guillaume Van Niel, Roosmarijn E. Vandenbroucke, Frederik J. Verweij, Ana V. Villar, Marca Wauben, Ann M. Wehman, Hang Yin, David Raul Francisco Carter, Pieter Vader
Another strategy for the labelling of vesicles is via fluorescent tagging of EV proteins. For example, tagging of tetraspanin proteins such as CD63 or CD81 with fluorescent proteins such as GFP or mCherry allows EVs to be visualized and tracked. Innovative approaches in which pH-dependent tags are employed, have been used to visualize EVs following fusion of the MVB with the PM [146]. This approach depends on the acidic environment of the multivesicular body and a neutral environment outside the cell. Alternatively, tagging abundant fluorescent reporters with a degradation motif (degron) is a way to specifically label EVs and remove the reporter from the source cell, allowing the observation of autocrine interactions in vivo [35,59,147]. Issues that need to be considered for any protein-tagging approach include the half-life of the fluorescent tag, the brightness of the tag, the limits of resolution of the microscopy technique used, and the possibility that tagged EVs (or indeed dye-labelled EVs) may have altered cargo or function. Another issue is that the visualization of EVs that lack the fluorescently tagged protein would be “invisible” and would thus be missing from any analysis. In this respect, general EV membrane labelling using, for example, fluorescent proteins fused to farnesylation or palmitoylation signals may be preferred [148,149]. It is recommended that further work is undertaken to optimize and establish the best methodology for the fluorescent tagging or labelling of EVs.