For liquid overlay 3D multicellular tumor spheroid formation, 96-well plates were coated with 0.15% agarose dissolved in PBS with 1% penicillin/streptomycin. One thousand UM-SCC-74B cells/well or 1500 UM-SCC-74A cells/well were seeded and incubated at 37°C in supplemented media for 3 days prior to irradiation with 2 Gy or mock radiation (0 Gy) using 137Cs gamma-ray photons as described above. Spheroid images were obtained at start of treatment and 10 days after treatment using a Canon EOS 700D camera mounted on an inverted Nikon Diaphot-TMD microscope. The images were analyzed using ImageJ version 1.48 (NIH, Bethesda, MD, USA), by measuring the surface area of each spheroid and calculating the volume, assuming each spheroid retained a spherical form. Each spheroid was normalized to its own starting volume at the start of treatment (Day 0, growth ratio = 1). Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Differences in normalized spheroid growth ratios of UM-SCC-74A cells vs. UM-SCC-74B cells were assessed using an unpaired t-test and were considered statistically significant if P < 0.05.
Diaphot tmd microscope
Manufactured by Nikon
Sourced in Japan
The Diaphot-TMD microscope is a laboratory equipment designed for various microscopy applications. It features a transmitted light illumination system and supports multiple observation methods, including phase contrast and dark field. The Diaphot-TMD microscope is capable of magnifying specimens for detailed examination and analysis.
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6 protocols using diaphot tmd microscope
1
Multicellular Tumor Spheroid Irradiation
2
Sorafenib and Onalespib Impact on Cell Migration
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Wound healing scratch assays were performed to investigate the migratory capacity of BHT-101, SW1736, 8305C and MDA-T32 treated with sorafenib and onalespib. Cells were grown in 6 well plates to 90% confluence and incubated with sorafenib (15 μM) and onalespib (50 and 100 nM) for 24 h. Then, a scratch was made in the cell monolayer using a pink (10 μL) pipette tip, followed by media rinse and incubation with fresh cell culture media. Wound closure was monitored and photographed at 6, 12, and 24 h using a Canon EOS 700D digital camera (Canon Inc., Japan) mounted on an inverted Nikon Diaphot-TMD microscope. Migration distance was measured and analyzed using ImageJ 2.0.0 software (NIH, Bethesda, MD, USA).
3
Immunolabeling of NF-κB and VCAM-1 in Tissue
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A rabbit antibody against the C-terminus of rat NF-kB p65 subunit [Rel A] was obtained from Millipore International Inc. (Amsterdam, Netherlands) (AB1604b), and goat anti-rat VCAM-1 (C-19) antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz USA) (sc-1504). Tissue sections were cut at a 3-µm thickness from paraffin-embedded blocks. The antibodies were diluted 1∶1000. The primary incubations were carried out for 1 hour at 21–22°C, followed by extensive six washes in PBS with Triton X-100 for 5 min each. The secondary antibodies, anti-rabbit IgG TR and anti-goat IgG FITC (Sigma-Aldrich), were diluted in PBS alone according to the manufacturer’s instructions.
The images were collected with Nikon EZ-C1 3.00 software on a Nikon Diaphot TMD microscope.
The images were collected with Nikon EZ-C1 3.00 software on a Nikon Diaphot TMD microscope.
4
Visualizing Apoptosis Stages via Annexin V
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To investigate the formation of apoptotic bodies and malformed cells, STS-induced cells were mounted on glass slides covered by cover slips and sealed with freshly opened nail polish. The changes in the nucleus, as well as cell shrinkage and deformities were observed under 1000 × magnification (Customized Nikon Diaphot TMD microscope). The progress of apoptosis within different phases from early apoptosis to necrosis was investigated by colouring the cells with annexinV conjugate Ex/Em 650/668 (Life technologies, Darmstadt, Germany). To label the cells with annexinV, the washed cells were resuspended in annexin-binding buffer {140 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher, Germany) (, 2.5 mM CaCl2, pH 7.4)}. 10 μl of annexinV conjugate was added to each well and it was incubated for 15 min at room temperature (RT). Thereafter, 10 μM of propidium iodide (PI): Ex/Em 533/617 nm (Thermo Fisher, Germany) was added to each well to assess apoptosis (AnnexinV+ PI−) and cell death (AnnexinV+ PI+). Cells were washed and injected into the micro channel and the signal variation from each apoptosis stage was detected hyperspectrally.
5
3D Spheroid Formation and Analysis
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For liquid overlay 3D spheroid formation, 96-well plates were coated with 0.15% agarose dissolved in PBS with 1% penicillin/streptomycin. 1000 UM-SCC-74B cells/well were seeded and incubated at 37°C for 3 days prior to treatment. The standard dose of 20 μM of PM2 was added 3 h after irradiation. Half of the medium was replaced every 48 h for the first 10 days, thereafter every 4 days. Samples with repeated PM2 treatments received a new 20 μM dose despite removing of half of the incubation medium. Images were obtained every 2–4 days using a Canon EOS 700D camera mounted on an inverted Nikon Diaphot-TMD microscope. The images were analyzed using ImageJ version 1.48 (NIH, Bethesda, MD, USA), by measuring the surface area of each spheroid and calculating the volume, assuming each spheroid retained a spherical form. Each spheroid was normalized to its own starting volume at the start of treatment (Day 0, growth ratio = 1). Spheroids exceeding a volume of 600 μm3 were excluded (i.e., terminated) from further analyses as 600 μm3 is the maximum size possible to obtain in these settings with uncompromised growth conditions.
6
Fluorescence Imaging of Differentiated PC12 Cells
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