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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.
Infertility Diagnosis and Treatment
Published in Sujoy K. Guba, Bioengineering in Reproductive Medicine, 2020
A human observer examining the semen microscopic image can readily identify spermatozoa and trace its contour. Also cells or other structures which are not spermatozoa can be distinguished. Building into an automated system such capability requires laying down in detail a set of rules for image processing. Different rules have been proposed and the parameters examined also vary amongst systems currently in use. A proven approach is based upon size, luminosity and motion determination. In part the discrimination can be done at the sample preparation stage itself where by using filters, objects definitely larger in size than spermatozoa can be filtered out. Further size discrimination is introduced in the computer software. Use of the phase contrast microscope helps too. Spermatozoa are luminous in phase contrast microscopy. Some other structures are either not luminous or significantly less luminous and in the gray scale image analysis a threshold can be set so that the computer ignores these objects. Live spermatozoa have velocities which lie in a certain range and objects with velocities beyond the range can be ignored.
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.
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).
Establishment of a reference procedure to measure urine-formed elements and evaluation of an automated urine analyzer
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2019
Rui Zhang, Huaian Ma, Huimin Yuan, Hongyan Guo, Bingxin Jiao, Yan Zhang, Xi Zhang, Huidong Dou, Zhiqi Gao, Qingtao Wang
The three main factors that may reduce the accuracy of a cell counting board are that it requires technical proficiency, there may be uneven distributions of components in the sample, and there may be identification errors [14]. An automated instrument provides an analysis that is more similar to bright-field than phase-contrast microscopy. Thus, we used bright-field microscopy in this study. Each of the Uric-SCP chamber plates used in this study has 16 quadrants and a volume of 1 µL. The use of too many or too few cells can affect the accuracy and precision of the counts; use of too many cells can cause an uneven distribution of cells, and use of too few cells reduces precision. In our experience, when sECs were counted at low magnification, there should be 50 to 80 cells/µL; when RBCs and WBCs are counted at high magnification, there should be 200 to 300 cells/µL. Satisfaction of these conditions provides good repeatability and precision.