All of the data acquisition is performed using optical video-microscopy set-ups. Experiments described in Chapter 3 to Chapter 6 utilise an Olympus inverted bright-field microscope while those in Chapter 7 use a custom-built inverted bright field microscope instead which allows for the set-up to be tilted to a small angle. Objectives with magnification of 20⇥ and 40⇥ are used
Inverted bright field microscope
Manufactured by Olympus
Sourced in Japan
The Inverted Bright Field Microscope is a versatile laboratory instrument designed for observation and analysis of samples. It features an upright optical path, allowing for easy access and manipulation of specimens. The microscope provides bright field illumination, enabling clear visualization of samples.
Automatically generated - may contain errors
Lab products found in correlation
Sa β gal, by Olympus (2 mentions)
Fluorescence microscopy, by Olympus (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Transwell insert, by Corning (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
Angiogenesis slides 15 well, by Ibidi (1 mentions)
Matrigel, by BD (1 mentions)
12 protocols using inverted bright field microscope
1
Optical Video-Microscopy Protocols
2
Hypoxia-Induced Cell Migration
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U87 and U251 cells (2×104 cells/well) were added to the upper chamber of the Transwell insert (Corning Incorporated), which contained serum-free DMEM. Medium containing 10% FBS was added to lower chamber as an attractant. Subsequent to incubation for 24 h in 1% O2, images of the migrated cells were captured by an inverted bright-field microscope (Olympus Corporation) and the results were quantified by counting the number of cells.
3
Progesterone, Mifepristone, and rTs-MAPRC2-Ab Assay
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The selected concentration of progesterone (P30 ng/mL), mifepristone (M100 ng/mL), and rTs-MAPRC2-Ab (rat antisera against rTs-MAPRC2) ratio (1:300 dilution) were used at F-AL and ML stages with both controls (only RPMI and EtOH-RPMI), collected as described above, and the previously described procedures were followed according to [23 (link),24 (link)]. The 2000 worms/well of both stages (F-AL, ML) were cultured in a 6-well plate, and a 6-well precondition was used. The cultured medium consists of RPMI-1640, 10% heat-inactivated FBS (Fetal bovine serum), and 2% antibiotics (100 U/mL penicillin; 100 mg/mL streptomycin) (Gibco, Paisley, UK), and incubated at 37 °C and 5% CO2 for 48 h and the medium changed after 24 h for all treatments. The female (F-AL) and male (M-AL) adult worms were separated under light microscopy using Axiovert Zeiss Microscope (25× Neo Plan objectives) and observed the phenotypic appearance at both stages (F-AL and ML) using an inverted bright field microscope (Olympus, Shibuya, Japan). In the F-AL stage, the pregnancy maintenance or aborted to NBL were observed. While at the ML stage ecdysis (molting process) and motility of parasites were noted.
4
Muscle Larvae SiRNA Knockdown Assay
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In 12-well culture plates (500 ML/500 μL) at 37 °C with 5% CO2, muscle larvae were cultured in RPMI-1640 (supplemented with penicillin 500 units/mL and streptomycin 500 mg/mL) with siRNA-180, siRNA-419, siRNA-559, siRNA-Control, and PBS control as described above [42 (link)]. ML stage parasites were tested for motility and ecdysis (molting process). An inverted bright field microscope (Olympus, Shibuya, Japan) was used to examine the phenotypic appearance [24 (link)].
5
Quantifying β-Galactosidase in BMMSCs
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First, untreated BMMSCs and treated BMMSCs were both fixed with 0.5% glutaraldehyde. After being washed with PBS, the cells were stained in X-gal solution overnight at 37 °C. Subsequently, all stained cells were observed with an inverted bright field microscope (Olympus, Tokyo, Japan), and approximately 500 cells were counted in random fields to determine the percentage of blue-stained β-galactosidase positive cells.
6
Hypoxia-Induced Angiogenesis Assay
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Each well of 15-well angiogenesis slides (ibidi GmbH, Martinsried, Germany) was supplemented with 10 µl Matrigel (BD Biosciences, San Jose, CA, USA) and incubated at 37°C in 1% O2 for 30 min. DMEM containing 10% FBS was used to culture hypoxia-induced U87 and U251 cells (24 h in 1% O2, 5% CO2 and 94% N2 at 37°C) that were or were not transfected with miR-576-3p/miR-NC, hsa-HIF-1α/Ctrl and shHIF-1α/shCtrl, separately or together. Subsequently, the supernatants of cultured U87 and U251 cells were collected and used to suspend HUVECs (1×104 cells/well), which were incubated for 9 h at 37°C in an atmosphere containing 1% O2. Tube formation was analyzed under an inverted bright-field microscope (Olympus Corporation).
7
Evaluating rTs-MAPRC2-Ab Efficacy on Nematode Stages
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At the ML and NBL stages, the selected concentration rTs-MAPRC2-Ab ratios (1:50, 1:200, and 1:800 dilutions) were used in conjunction with controls pET32a rat serum (control group) and PBS (control group). A 6-well plate was used with 2000 worms/well of both stages (ML, NBL). Culture medium was composed of RPMI-1640, 10% heat-inactivated FBS (Fetal Bovine Serum), and 2% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (Gibco, Paisley, UK), and incubated at 37 °C with 5% CO2. ML stage parasites were tested at various time intervals (0 h, 4 h, 8 h, 24 h) for motility and ecdysis (molting process) following methods in the literature [33 (link),34 (link),35 (link)]. NBL motility was checked after 24 h. An inverted bright field microscope (Olympus, Shibuya, Japan) was used to examine the phenotypic appearance at both stages (ML and NBL) [24 (link)].
8
Wound Healing Assay in U87 and U251 Cells
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U87 and U251 cells were seeded in 6-well plates at a density of 1×104 cells/well and grown until complete fusion. The cell layers were scratched using a 200-µl pipette tip to generate a linear wound. Phosphate buffered saline was used to remove the previous DMEM and then serum-free DMEM was added. Images of the migrated cells were captured at 0 and 24 h through an inverted bright-field microscope (Olympus Corporation, Tokyo, Japan). Each experiment was performed three times.
9
Senescence-Associated β-Galactosidase Microscopy
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For senescence associated β-galactosidase (SA-β-gal) detection microscopy analysis was performed with the inverted bright field microscope (Olympus). Fluorescence signals were detected by fluorescence microscopy (Olympus).
For statistical analysis student's t test was performed.
For statistical analysis student's t test was performed.
10
Intracellular Iron Visualization by Prussian Blue
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Prussian blue staining was used to confirm the presence of intracellular iron. Briefly, SS treated cells were washed 3× with PBS and fixed using 4% formaldehyde solution for 30 min. Prussian blue staining (10 mg/mL) was done by incubating the fixed cells for 30 min and then rewashed 3× with PBS. Stained cells were examined under an inverted bright field microscope (Olympus, Tokyo, Japan) to determine intracellular iron distribution.
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