scholarly journals Delivery of Foreign Materials into Adherent Cells by Gold Nanoparticle-Mediated Photoporation

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 550
Author(s):  
Xiaofan Du ◽  
Jing Wang ◽  
Lan Chen ◽  
Zhenxi Zhang ◽  
Cuiping Yao
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Adherent Cells ◽  
Thermal Dynamics ◽  
Cervical Cell ◽  
Nanosecond Pulse Laser ◽  
Fluorescein Isothiocyanate Dextran ◽  
Extracellular Materials ◽  
Foreign Materials ◽  
Cell Medium ◽  
Delivery Efficiency

Delivering extracellular materials into adherent cells presents several challenges. A homemade photoporation platform, mediated by gold nanoparticles (AuNPs), was constructed to find a suitable method for finding all adherent cells in this process with high delivery efficiency. The thermal dynamics of AuNPs could be monitored. Based on this system, 60 nm AuNPs were selected to be attached to cells for optimal photoporation. After irradiating the cells covered with AuNPs using a nanosecond pulse laser, fluorescein isothiocyanate-dextran in the medium were delivered into optoporated adherent HeLa (human cervical cell lines) cells. The delivery efficiency and cell viability of this process were evaluated using a fluorescence microscope and flow cytometry. The experimental results showed that targeting cells using antibodies, laser irradiation from the top of the cell culture well, and reducing the cell medium are important for improving the delivery efficiency. The optimal loading efficiency for adherent HeLa cells was 53.4%.

scholarly journals Spray-Dried Plasma Improves Body Weight, Intestinal Barrier Function, and Tibia Strength during Experimental Constant Heat Stress Conditions

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2213
Author(s):  
Jared Ruff ◽  
Thaina L. Barros ◽  
Joy Campbell ◽  
Ricardo González-Esquerra ◽  
Christine N. Vuong ◽  
...  
Keyword(s):  
Heat Stress ◽  
Intestinal Barrier ◽  
Fluorescein Isothiocyanate ◽  
Serum Levels ◽  
Negative Control ◽  
Intestinal Barrier Function ◽  
Negative Consequences ◽  
Fluorescein Isothiocyanate Dextran ◽  
Prolonged Heat ◽  
Spray Dried

The aim of this study was to see how spray-dried plasma (SDP) supplementation affected broiler chicken performance, intestinal permeability, and bone strength during persistent heat stress. One-day-old chicks (n = 480) were randomly assigned into twelve environmental corrals; four thermoneutral (TN-negative control, maintained at 24 °C from d 21–42); four heat stress (HS, exposed to 35 °C from d 21–42); and four heat stress treated with 2% SDP in the feed until d 28 followed by 1% SDP until d 42 (HS-SDP). The performance and serum levels of fluorescein isothiocyanate-dextran (FITC-d) were evaluated at d 21, 28, 35, and 42. The tibias strength was evaluated on d 21 and 42. The increment in chicken temperature (p < 0.05) was observed two h following the increase in environmental temperature in both HS groups and was associated with decreased performance parameters compared with the TN group. At d 42 of age, the chickens exposed to HS had an impaired gut permeability and decreased tibia strength compared to the TN group (p < 0.05). However, partially feeding SDP mitigated these adverse effects significantly. These findings imply that using SDP strategically during stressful times, such as prolonged heat stress, may help mitigate its negative consequences.

Spreading of microinjected horseradish peroxidase to nondescendant cells in embryos of Patella (Mollusca, Gastropoda)

Development ◽  
1987 ◽  
Vol 100 (4) ◽  
pp. 713-722
Author(s):  
W.M. Kuhtreiber ◽  
F. Serras ◽  
J.A.M. van den Biggelaar
Keyword(s):  
Molecular Weight ◽  
Stem Cell ◽  
Horseradish Peroxidase ◽  
High Molecular Weight ◽  
Fluorescein Isothiocyanate ◽  
Contact Phase ◽  
High Molecular Weight Form ◽  
Contact Stage ◽  
Fluorescein Isothiocyanate Dextran ◽  
Molecular Weight Form

We have injected horseradish peroxidase (HRP) and fluorescein-isothiocyanate dextran (FD) into cells and into the blastocoelic cavity of Patella vulgata embryos, before and during the interval between 5th and 6th cleavage, in which the mesodermal stem cell is determined by means of interactions between the central 3D macromere and the contacting animal micromeres. Intracellular injections of HRP at different stages showed that, whereas before this contact phase no spreading of label was observed, a clear intercellular transfer of HRP was found after the contact was established. Control experiments showed that it was HRP in its intact, high molecular weight form that was transferred in the living embryo. Injections of HRP into the blastocoelic cavity gave essentially the same results. In these cases, the HRP was taken up by the cells from contact stage onwards. When FD was injected into the blastocoelic cavity, no uptake was observed, not even after prolonged presence of FD in it. However, when HRP and FD were mixed, both were taken up, starting at contact stage. Differences in labelling pattern of HRP, as compared with FD, and a shift of the FD fluorescence after uptake, suggest that receptor-mediated endocytosis is involved. The possible morphogenetic significance of the transfer mechanism is discussed.

The Effects of External Salinity on the Drinking Rates of the Larvae of Herring, Plaice and Cod

1988 ◽  
Vol 138 (1) ◽  
pp. 1-15 ◽  
Author(s):  
P. TYTLER ◽  
J. H. BLAXTER
Keyword(s):  
Time Course ◽  
Gadus Morhua ◽  
Sea Water ◽  
Fluorescein Isothiocyanate ◽  
Yolk Sac ◽  
Clupea Harengus ◽  
Larval Stages ◽  
First Feeding ◽  
Fluorescein Isothiocyanate Dextran ◽  
External Salinity

Drinking responses to salinity change in the larvae of herring (Clupea harengus L.), plaice (Pleuronectes platessa L.) and cod (Gadus morhua L.) were measured from the time course of uptake of dextran labelled with tritium, following immersion in solutions of 32‰ and 16‰ sea water. The yolk sac and first feeding larval stages of all three species drink in both salinities. Furthermore, post-yolk sac stages appear to adjust their drinking rates to compensate for different salinities in a manner similar to that of the adults. Drinking rates in 32‰ sea water are approximately double those in 16‰. Mass-related drinking rates of larvae are higher than those in adults, but the differences do not match the differences in surface area to mass ratios, suggesting that larval skin is less permeable to water than is adult gill epithelium. Water absorption is indicated by the evidence of concentration of dextran in the gut. The estimates of drinking rates from tritiated dextran uptake are supported by epifluorescence microscopical measurements of the uptake of fluorescein isothiocyanate dextran.

NK1 receptors mediate tachykinin-induced increase in microvascular clearance in hamster cheek pouch

1993 ◽  
Vol 265 (2) ◽  
pp. H593-H598
Author(s):  
X. P. Gao ◽  
R. A. Robbins ◽  
R. M. Snider ◽  
J. Lowe ◽  
S. I. Rennard ◽  
...  
Keyword(s):  
Substance P ◽  
Fluorescein Isothiocyanate ◽  
Cheek Pouch ◽  
Neurokinin A ◽  
Plasma Extravasation ◽  
Hamster Cheek Pouch ◽  
Neurokinin B ◽  
Dextran 70 ◽  
Fluorescein Isothiocyanate Dextran ◽  
Neurogenic Plasma Extravasation

The purpose of this study was to determine the receptor subtype(s) that mediates tachykinin-induced neurogenic plasma extravasation in the hamster cheek pouch. Changes in microvascular clearance were quantified by counting the number of leaky sites and calculating the clearance of fluorescein isothiocyanate-dextran [mol wt 70,000 (Dextran 70)] during suffusion of the cheek pouch with substance P, neurokinin A, neurokinin B, and capsaicin. Suffusion of substance P, capsaicin, and neurokinin A, but not neurokinin B, was associated with a significant concentration-dependent increase in leaky site formation and clearance of fluorescein isothiocyanate-Dextran 70 (P < 0.05). However, the responses to substance P and capsaicin were significantly greater than those to neurokinin A. Pretreatment with the selective, nonpeptide NK1 receptor antagonist, CP-96,345, significantly attenuated substance P- and capsaicin-induced but not neurokinin A-induced responses (P < 0.05). These effects were specific, since the 2R,3R enantiomer, CP-96,344, was inactive, and CP-96,345 had no significant effect on adenosine-induced responses. We conclude that, in the hamster cheek pouch, NK1 receptors are the predominant receptors that mediate neurogenic plasma extravasation.

HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers

2006 ◽  
Vol 290 (4) ◽  
pp. C990-C999 ◽  
Author(s):  
Shiguang Liu ◽  
Donna B. Stolz ◽  
Penny L. Sappington ◽  
Carlos A. Macias ◽  
Meaghan E. Killeen ◽  
...  
Keyword(s):  
High Mobility ◽  
Fluorescein Isothiocyanate ◽  
Intestinal Epithelial ◽  
Average Molecular Mass ◽  
Diffusion Chambers ◽  
Primary Mouse ◽  
Tlr4 Agonist ◽  
Ifn Γ ◽  
Fluorescein Isothiocyanate Dextran ◽  
Culture Supernatants

High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with “cytomix” (a mixture of TNF, IL-1β, and IFN-γ) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.

scholarly journals Macromolecular Capillary Leakage Is Involved in the Onset of Anaphylactic Hypotension

2012 ◽  
Vol 117 (5) ◽  
pp. 1072-1079 ◽  
Author(s):  
Nathalie Faye ◽  
Laure Fournier ◽  
Daniel Balvay ◽  
Rokhaya Thiam ◽  
Gilles Orliaguet ◽  
...  
Keyword(s):  
Signal Intensity ◽  
Anaphylactic Shock ◽  
Fluorescein Isothiocyanate ◽  
Brown Norway ◽  
Intensity Increase ◽  
Shock Onset ◽  
Capillary Leakage ◽  
Microscopy Imaging ◽  
Arterial Blood ◽  
Fluorescein Isothiocyanate Dextran

Background The role of the hypovolemic component secondary to the microcirculatory changes in the onset of inaugural anaphylactic hypotension remains debated. We investigated the microcirculatory permeability in a model of anaphylactic shock using a fluorescence confocal microscopy imaging system. Methods Ovalbumin-sensitized anesthetized Brown Norway rats were randomly allocated into two groups (n = 6/group): control and anaphylaxis, respectively induced by intravenous saline or ovalbumin at time 0 (T0). The mesentery was surgically exposed. Macromolecular fluorescein isothiocyanate-dextran was intravenously injected (T0-5min) allowing in vivo visualization of the mesenteric microvascular network by fluorescence microscopy. After a period of stabilization of the contrast agent concentration, a 5-s movie was recorded to obtain baseline signal intensity. Following T0, 5-s movies were recorded every 30 s for 30 min. Capillary leakage of fluorescein isothiocyanate-dextran was assessed in interstitium and compared between groups. Data are expressed as mean ± SD. Results Following anaphylactic shock onset, an early, progressive, and global signal intensity increase over time was detected in the interstitium. Mean index leakage differed between control and anaphylaxis (respectively 20 ± 11 vs. 170 ± 127%; P &lt; 0.0001), starting at 2 min after shock onset and progressively increasing. Index leakage correlated with the drop in arterial blood pressure until T0 + 10 min (r = -0.75, P = 0.0001). Conclusions During anaphylaxis, interstitial capillary leakage occurs within minutes after shock onset. Compared with controls, the mesenteric microcirculation showed at least 8-fold-increased macromolecular capillary leakage. The inflammation-induced microcirculatory changes with subsequent intravascular fluid transfer might be involved in the onset of the inaugural hypotension during anaphylactic shock.

scholarly journals Polysaccharide Hydrogels Support the Long-Term Viability of Encapsulated Human Mesenchymal Stem Cells and Their Ability to Secrete Immunomodulatory Factors

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Fahd Hached ◽  
Claire Vinatier ◽  
Pierre-Gabriel Pinta ◽  
Philippe Hulin ◽  
Catherine Le Visage ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hydroxypropyl Methylcellulose ◽  
Human Mesenchymal Stem Cells ◽  
Fluorescein Isothiocyanate ◽  
Spherical Particles ◽  
Dispersion Method ◽  
Fluorescein Isothiocyanate Dextran ◽  
Average Size ◽  
Stimulation Of

While therapeutically interesting, the injection of MSCs suffers major limitations including cell death upon injection and a massive leakage outside the injection site. We proposed to entrap MSCs within spherical particles derived from alginate, as a control, or from silanized hydroxypropyl methylcellulose (Si-HPMC). We developed water in an oil dispersion method to produce small Si-HPMC particles with an average size of about 68 μm. We evidenced a faster diffusion of fluorescein isothiocyanate-dextran in Si-HPMC particles than in alginate ones. Human adipose-derived MSCs (hASC) were encapsulated either in alginate or in Si-HPMC, and the cellularized particles were cultured for up to 1 month. Both alginate and Si-HPMC particles supported cell survival, and the average number of encapsulated hASC per alginate and Si-HPMC particle (7102 and 5100, resp.) did not significantly change. The stimulation of encapsulated hASC with proinflammatory cytokines resulted in the production of IDO, PGE2, and HGF whose concentration was always higher when cells were encapsulated in Si-HPMC particles than in alginate ones. We have demonstrated that Si-HPMC and alginate particles support hASC viability and the maintenance of their ability to secrete therapeutic factors.

Role of tumor necrosis factor–α and matrix metalloproteinase–9 in blood-brain barrier disruption after peripheral thermal injury in rats

2009 ◽  
Vol 110 (6) ◽  
pp. 1218-1226 ◽  
Author(s):  
Raul Reyes ◽  
Miao Guo ◽  
Kathryn Swann ◽  
Siddharth U. Shetgeri ◽  
Shane M. Sprague ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Expression ◽  
Tumor Necrosis ◽  
Thermal Injury ◽  
Fluorescein Isothiocyanate ◽  
Tnf Α ◽  
Fluorescein Isothiocyanate Dextran ◽  
Factor Α ◽  
Metalloproteinase 9 ◽  
Necrosis Factor

Object A relationship has been found between peripheral thermal injury and cerebral complications leading to injury and death. In the present study, the authors examined whether tumor necrosis factor–α (TNF-α) and matrix metalloproteinase–9 (MMP-9) play a causative role in blood-brain barrier (BBB) disruption after peripheral thermal injury. Methods Thirty-two male Sprague-Dawley rats were subjected to thermal injury. One hour later, 8 rats were injected with TNF-α neutralizing antibody, and 8 were injected with doxycycline, an inhibitor of the MMP family proteins; 16 rats did not receive any treatment. Brain tissue samples obtained 7 hours after injury in the treated animals were examined for BBB function by using fluorescein isothiocyanate–dextran and by assessing parenchymal water content. Protein expression of basement membrane components (collagen IV, laminin, and fibronectin) was quantified on Western blot analysis, and MMP-9 protein expression and enzyme activity were determined using Western blot and gelatin zymography. Thermally injured rats that did not receive treatment were killed at 3, 7, or 24 hours after injury and tested for BBB functioning at each time point. Histological analysis for basement membrane proteins was also conducted in untreated rats killed at 7 hours after injury. Results of testing in injured rats were compared with those obtained in a control group of rats that did not undergo thermal injury. Results At 7 hours after thermal injury, a significant increase in the fluorescein isothiocyanate–dextran and water content of the brain was found (p < 0.05), but BBB dysfunction was significantly decreased in the rats that received TNF-α antibody or doxycycline (p < 0.05). In addition, the components of the basal lamina were significantly decreased at 7 hours after thermal injury (p < 0.01), and there were significant increases in MMP-9 protein expression and enzyme activity (p < 0.05). The basal lamina damage was reversed by inhibition of TNF-α and MMP-9, and the increase in MMP-9 protein was reduced in the presence of doxycycline (p < 0.05). The authors found that MMP-9 enzyme activity was significantly increased after thermal injury (p < 0.01) but decreased in the presence of either TNF-α antibody or doxycycline (p < 0.01). Conclusions The dual, inhibitory activity of both TNF-α and MMP-9 in brain injury suggests that a TNF-α and MMP-9 cascade may play a key role in BBB disruption. These results offer a better understanding of the pathophysiology of burn injuries, which may open new avenues for burn treatment beyond the level of current therapies.

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