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Eosinophils in Immunological Reactions
Published in Gerald J. Gleich, A. Barry Kay, Eosinophils in Allergy and Inflammation, 2019
The solubility of lipid bodies in conventional alcohol-based hematological stains, e.g., Wright’s and Giemsa stains, causes lipid bodies to be dissolved and missed. In contrast, if lipid is first preserved by exposure to osmium-tetroxide prior to staining or if staining is effected with the lipophilic fluorescent dye Nile red, then lipid body accumulations within leukocytes can be recognized and enumerated. With such specific staining, lipid body numbers can be recognized by light or electron microscopy to be increased in leukocytes associated with various inflammatory and immunological reactions (11). Eosinophil lipid bodies have an ultrastructural morphology similar to those found within neutrophils but are characteristically more electron dense following osmium staining, like lipid bodies in mast cells (12,13,15). Whether the heightened osmiophilic nature of eosinophil lipid bodies reflects preferential associations of specific types or quantities of osmiophilic phospholipids or proteins with these structures is not known. While lipid bodies lack a delimiting membrane, they often possess a more electron-dense peripheral shell and are found enmeshed in cytoskeletal elements (14). Eosinophil lipid bodies, with their dark osmiophilic staining, their comparable size to granules, and their heightened numbers in activated eosinophils, have at times been misidentified as cytoplasmic granules.
Advanced Formulation Techniques Including Innovative Materials
Published in Heather A.E. Benson, Michael S. Roberts, Vânia Rodrigues Leite-Silva, Kenneth A. Walters, Cosmetic Formulation, 2019
Bozena Michniak-Kohn, Tannaz Ramezanli, Frank Romanski, Cliff Milow, Kishore Shah
Polymeric nanoparticulate–based drug delivery systems have been investigated by several groups for the treatment of dermatological diseases (Zhang et al., 2013). The rationale is that these nanoparticles can protect the drug from degradation, provide sustained/controlled release of their cargo, and improve partitioning and permeation into the skin. To evaluate the potential use of TyroSpheres as a skin delivery system, fluorescent dyes, for example, Nile red (log D: 3.10) and 5-dodecanoylaminofluorescein (DAF, logD: 7.54), were loaded into nanospheres separately, and their percutaneous penetration using human cadaver skin was assessed and compared to controls comprising solutions in propylene glycol. The studies were conducted using vertical Franz diffusion cells. It was found that Nile red and DAF delivery to skin strata was 9.0 and 2.5 times enhanced, respectively, when formulated into TyroSpheres relative to propylene glycol (Sheihet et al., 2008). Moreover, viscous formulations of TyroSpheres – that are more suitable than liquid dispersions for topical applications – were prepared using hydroxypropyl methylcellulose (HPMC) and propylene glycol. In an ex vivo study on human cadaver skin, the viscous formulations of Nile red TyroSpheres resulted in similar dye deposition in the stratum corneum and viable epidermis compared to a TyroSphere liquid dispersion. Remarkably, in an in vivo study on pigs, topically applied Nile red–loaded TyroSphere in a viscous formulation resulted in 40% higher dye delivery to skin than that obtained from Nile red–TyroSphere liquid dispersion (applied via Hill Top Chambers® in an occlusive patch test system) (Batheja et al., 2011).
PEGylated Dendritic Nanoparticulate Carriers of Anti-Cancer Drugs
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
D. Bhadra, S. Bhadra, N. K. Jain
Gillies, Jonsson, and Frechet (2004) used a pH-responsive micelle system, linear-dendritic block copolymers comprising PEO and either a polylysine or polyester dendron. These were prepared, and hydrophobic groups were attached to the dendrimer periphery by highly acid-sensitive cyclic acetals. These copolymers were designed to form stable micelles in aqueous solution at neutral pH and disintegrate into unimers at mildly acidic pH following loss of the hydrophobic groups upon acetal hydrolysis. Micelle formation was demonstrated by encapsulation of the fluorescent probe Nile Red, and the micelle sizes were determined by dynamic light scattering. The structure of the dendrimer block, its generation, and the synthetic method for linking the acetal groups to its periphery all had an influence on the CMC and the micelle size. The rate of hydrolysis of the acetals at the micelle core was measured for each system at pH 7.4 and pH 5, and it was found that all systems were stable at neutral pH but that they underwent significant hydrolysis at pH 5 over several hours. The rate of hydrolysis at pH 5 was dependent on the structure of the copolymer, most notably the hydrophobicity of the core-forming block. To demonstrate the potential of these systems for controlled release, the release of Nile Red as a model payload was examined. At pH 7.4, the fluorescence of micelle-encapsulated Nile Red was relatively constant, indicating it was retained in the micelle, and at pH 5, the fluorescence decreased, consistent with its release into the aqueous environment. The rate of release was strongly correlated with the rate of acetal hydrolysis and was controlled by the chemical structure of the copolymer. The mechanism of Nile Red release was investigated by monitoring the change in size of the micelles over time at acidic pH. Dynamic light scattering measurement showed a size decrease over time, eventually reaching the size of a unimer, providing evidence for the proposed micelle disintegration.
Attenuation of obesity related inflammation in RAW 264.7 macrophages and 3T3-L1 adipocytes by varanadi kashayam and identification of potential bioactive molecules by UHPLC-Q-Orbitrap HRMS
Published in Archives of Physiology and Biochemistry, 2023
J. U. Chinchu, Mohind C. Mohan, B. Prakash Kumar
Intracellular lipid quantification was done by fluorescent Nile red staining as per the protocol described earlier (Chinchu et al.2019a). In brief, differentiation was induced in 3T3-L1 preadipocytes in the presence of 6.25, 25 and 100 µg/mL of Varanadi kashayam fractions. On the 10th day, after the induction of differentiation, the medium was replaced with RAW CM and incubated for 16 h. Thereafter, cells fixed with 4% formalin and stained with Nile red (5 µg/mL) and fluorescent intensity of lipid droplets measured at 485 excitation wavelength and 550 emission wavelength. The percentage of lipid droplets in 3T3-L1 cells measured by the formula mentioned in Wang et al. (2014). 100 × (RFUSample – RFUPreadipocyte)/(RFUControl – RFUPreadipocyte), where RFUPreadipocyte means relative fluorescence unit of undifferentiated stained cells; RFUControl means relative fluorescence unit of mature differentiated adipocytes and RFUSample means relative fluorescence unit of adipocytes treated with Varanadi kashayam fractions. The expression of adipogenic transcription factors in RAW CM stimulated 3T3-L1 adipocytes was quantified by quantitative real-time PCR. The sequence of primers used for qRT-PCR given in Table 1.
In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2021
Carlos E. Navarro Chica, Tian Qin, Bart J. de Haan, M.M Faas, Alexandra M. Smink, Ligia Sierra, Betty L. López, Paul de Vos
Labelled Sq-GusNPs were obtained as previously reported [2]. Briefly, an ethanolic solution of Nile Red at a concentration of 0.1 mg/mL was prepared. From this 380 µL was taken and ethanol was evaporated using the concentrator SpeedVac SPD2010 to obtain dried crystals of Nile Red. Subsequently, 380 µL of Sq-Gus bioconjugate dissolved in ethanol at a concentration of 2 mg/mL was added to the dried Nile Red crystals, vortexed, and NPs obtained as described in the previous section (Sq-GusNPs preparation). The aqueous suspension of labelled Sq-GusNPs was filtered through a 0.22 µm filter to separate the Nile red precipitate after ethanol evaporation obtaining a translucid suspension of labelled Sq-GusNPs. The prepared NPs had a size of 154.9 ± 47.0 nm as measured by DLS with the particle size analyzer NICOMP 380 ZLS.
Simple techniques to study multifaceted diabesity in the fly model
Published in Toxicology Mechanisms and Methods, 2019
Nibedita Nayak, Monalisa Mishra
These tissues can be dissected and stained by a vital dye, Nile red which is used to detect intracellular lipid droplet in unfixed tissues using fluorescence microscopy, where the neutral lipid is viewed as yellow-gold fluorescence (Greenspan et al. 1985; Grönke et al. 2005). The lipid droplets, both its size and number, reflect the status of fat deposition and state of lipolysis (Greenspan et al. 1985). Larva gut, fat body, and the imaginal disc can be dissected and kept in 4% paraformaldehyde for overnight. Next, the samples were washed with phosphate buffered saline (PBS). Next day, the samples were washed three times and the time interval of each wash is 5 min. Then, wash with PBS with 1% Twin-20 (PBST) for 2 times 5 min each wash. Stain the sample with Nile red (0.5 µg/ml) and incubate for 1 h. Wash the excess stain with PBS. Next, fix the samples with 20% glycerol and observe under a fluorescence/confocal microscope. Take the images from appropriate areas. The number and size of the lipid droplet are being counted and measured in Image J software (Liu et al. 2012). The stained images of the Drosophila tissue are represented in Figure 3.