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Alternative Methods for Assessing the Effects of Chemicals in the Eye
Published in David W. Hobson, Dermal and Ocular Toxicology, 2020
Leon H. Bruner, John Shadduck, Diane Essex-Sorlie
Selected technique — Highest tolerated dose.37,39–41 BALB/c 3T3 cells are seeded into 96-well culture plates at densities sufficient to obtain semiconfluent cultures 24 h after seeding. Growth medium is removed and replaced with medium containing the test chemicals; then, the cells are incubated in the presence of the chemical for 24 h. Cells are observed microscopically and evaluated for morphologic alterations using phase contrast microscopy. Various concentrations of the test chemical are used, and the highest concentration of the chemical that does not produce morphological alterations is designated the highest tolerated dose. Unexposed BALB/c 3T3 cells are used for comparison. The indices evaluated include decreased cell density, increased cell roundedness, and cellular cytoplasmic granularity. In terms of ranking a chemical relative to others in a set, agreement with other in vitro tests and rabbit eye irritation test data has been good in a limited number of evaluations.
Magnetic Control of Biogenic Micro-Mirror
Published in Shoogo Ueno, Bioimaging, 2020
For example, illumination of cells in the vicinity of guanine platelets was demonstrated in Figure 10.16. In that optical configuration, incident light came from the side of the chamber containing biogenic fish guanine platelets floating in water. The reflected light from the platelets illuminated osteoblast cells (MC3T3-E1); hence, the platelets acted as half mirrors. The cells were imaged much like in phase-contrast microscopy. On the left in Figure 10.16, the layer of guanine platelets and the cell layer are separated from each other. When cells were cultured with guanine platelets, the distance between the cells and the micro-mirrors can be reduced to micro-meters. Light illumination of cells at a short distance was demonstrated by placing a tiny biogenic reflecting platelet on a cultured cell layer (Figure 10.17). The results suggest possible applications of cellular imaging by the reflecting particles.
Influences of Hyperthermia on Immunological Functions
Published in Leopold J. Anghileri, Jacques Robert, Hyperthermia In Cancer Treatment, 2019
Marianne J. Skeen, John R. McLaren, Zbigniew L. Olkowski
Lymphocyte viability is routinely measured by exclusion of trypan blue dye. In our laboratory, lymphocytes isolated from normal human donors remained greater than 90% viable by trypan blue exclusion after 30 min of exposure to temperatures up to 47°C.1 Viability decreased to 78% after 30 min at 48.5°C, but remained as high as 48% after 30 min at 53.5°C. Viability was routinely tested immediately following heating, but limited testing after 18 hr further incubation at 37°C indicated no further cell death. Schrek2 investigated long-term viability by incubating isolated human lymphocytes at elevated temperatures for 2 to 200 min followed by a 7-day incubation at 37°C. Viability was determined using phase contrast microscopy. In comparison to unheated control cultures, cells heated at 43°C for 60 min showed only a 10% decrease in viability, whereas cells heated at 45°C for only 30 min were nearly 60% less viable than controls after 7 days. The important finding of these studies is that relatively brief exposure to temperatures as high as 43°C had only minimal effects on viability per se.
Effect of Long-term Anti-VEGF Treatment on Viability and Function of RPE Cells
Published in Current Eye Research, 2022
Anna Brinkmann, Katrin Winkelmann, Tom Käckenmeister, Johann Roider, Alexa Klettner
Porcine RPE cells were prepared as previously described.29 In brief, porcine eyes were obtained from a local abattoir, and the cornea, lens, vitreous, iris, ciliary body and retina were removed. RPE cells were harvested by trypsin digestion and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, PAA, Cölbe, Germany) supplemented with penicillin/streptomycin (1%), L-glutamine, amphotericine B (0.5 μg/ml), HEPES (25 mM), sodium-pyruvate (110 mg/ml) (all PAA) and 10% fetal calf serum (Linaris, Wertheim-Bettingen, Germany). Cells were cultivated in 12-well plates. For phagocytosis assays, cells were cultivated on collagen-coated (Collagen A, Biochrome, Berlin, Germany) cover slips (21x26 mm, Menzel GmbH, Braunschweig, Germany). For transepithelial electrical resistance (TER) measurements, cells were cultivated on transwell membranes (Sarstedt, #83.3931.041). Morphology of the cells was assayed with phase contrast microscopy. Cells were not passaged. Confluent cells displaying a characteristic RPE morphology were used for experimentation.
Comparison of Virosome vs. Liposome as drug delivery vehicle using HepG2 and CaCo2 cell lines
Published in Journal of Microencapsulation, 2021
Varun Kumar, Ramesh Kumar, V. K. Jain, Suman Nagpal
Differences in cell morphology have been observed between treated and controlled cells by phase-contrast microscope. The most conspicuous morphological changes were noticed in the case of NC-Virosome treated cells as compared to the controlled cells, involving irregular shape, shrinkage, and loss of attachment to the substratum; these observations support the characteristic features of cell death (Figure 6). The disintegrated cell membrane and blebbing of cells were observed in group-IA, -IIA & -IIIA and Group-IB, -IIB & -IIIB with 100 µg/ml of NC-Virosome, NC-Liposome and Curcumin after 48 h; respectively. Maximum damage to cell morphology was observed in the case of NC-Virosome-treated cells. In adherent cells, loss of attachment was associated with morphological changes characteristics of cell death such as blebbing, shrinkage, and condensation of the chromosome (Zhang et al. 2004, Vakifahmetoglu et al. 2008). In conclusion, the cells treated with NC-Virosome induced cell death by necrosis and favors anti-proliferation of cells.
Edaravone prevents high glucose-induced injury in retinal Müller cells through thioredoxin1 and the PGC-1α/NRF1/TFAM pathway
Published in Pharmaceutical Biology, 2021
Identification of Müller cells was performed after passaging the cells to the third generation. A cover glass was placed in a Petri dish. Next, the cells were digested and seeded on the cover glass. When the cells grew close to confluence, they were removed, washed with D-Hank’s solution, fixed with 4% paraformaldehyde (20 min) and treated with 0.1% Triton (15 min). The endogenous peroxidase was blocked by incubating the cells with 0.3% H2O2 for 20 min. After being blocked with normal goat serum (Beyotime, Shanghai, China) for 20 min, mouse anti-glial fibrillary acidic protein (GFAP, 1:200; Sigma-Aldrich, USA) or rabbit anti-glutamine synthetase (GS, 1:1,000; Sigma-Aldrich, USA) was added, while PBS (0.01 mol/L, pH 7.2) was added to the control group. The samples were incubated overnight at 4 °C and eluted with PBS. A goat anti-rat IgG-HRP was used as the secondary antibody (No. sc-2005, 1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 h under room temperature). Upon elution with PBS, Hoechst was added to stain the nucleus. Cells were observed under a fluorescence inverted phase contrast microscope. The immunofluorescence staining showed that the majority of cells were GFAP- and GS-positive, indicating the purity of cells. The cell bodies and protrusions emitted red fluorescence, while the nucleus was Hoechst-positive with blue fluorescence, indicating that these cells were Müller cells (>90% of cells were identified as Müller cells).