Fixed or living cells were observed at 25°C or 37°C with 5% CO2, respectively, under a TE2000 microscope (Nikon) equipped with a 100× 1.4 NA Plan-Apochromatic, a 60× 1.4 NA Plan-Apochromatic, or a 40× 1.3 NA Plan Fluor oil immersion objective lens (Nikon), a CSU-X1 confocal unit (Yokogawa), and an iXon3 electron multiplier-charge–coupled device camera (Andor) or an ORCA-ER CCD camera (Hamamatsu Photonics). Image acquisition was controlled by µManager software (Open Imaging). Long-term live imaging for cell cycle analyses was performed using a LCV110 microscope (Olympus) equipped with a 40× 0.95 NA UPLSAPO dry lens (Olympus). Because we found that 488-nm light irradiation severely interfered the progression of cell cycle and mitosis in HAP1 cells, we used bright-field microscope for long-term live imaging for cell cycle analyses (Figs. 1 , 8 , and S3). For live imaging, cells were cultured in phenol red–free IMDM (Thermo Fisher Scientific) supplemented with 10% FBS and 1× AA.
Lcv110 microscope
Manufactured by Olympus
Sourced in Japan
The LCV110 is a compact and versatile microscope designed for laboratory use. It features a binocular viewing head, LED illumination, and coaxial coarse and fine focusing controls. The microscope is suitable for a range of magnification levels and can be used for various applications in the life sciences and materials research fields.
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Lab products found in correlation
Te2000 microscope, by Nikon (1 mentions)
Plan fluor oil immersion objective lens, by Nikon (1 mentions)
Phenol red free imdm, by Thermo Fisher Scientific (1 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Plan apochromatic, by Nikon (1 mentions)
Csu x1 confocal unit, by Yokogawa (1 mentions)
Ixon3 electron multiplier charge coupled device camera, by Oxford Instruments (1 mentions)
Glass bottomed dish, by Iwaki (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
4 protocols using lcv110 microscope
1
Live Cell Imaging of Cell Cycle
2
Live-Cell Imaging of Histone H2B
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HeLa cells stably expressing GFP-tagged histone H2B were seeded onto a glass-bottomed dish (Iwaki). Time-lapse fluorescence microscopy and differential interference contrast video microscopy were performed using an Olympus LCV110 microscope. Images were acquired using a charge-coupled device camera (Retiga EXi, QImaging) equipped with a U Plan Super Apochromatic ×40, 0.95 numerical aperture objective, and then analyzed using MetaMorph software (Molecular Devices).
3
Live Cell Imaging of Dynamic d2EGFP Expression
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For live cell imaging, 8 × 104 MT1GFP cells were seeded in a 5-mm glass-bottom dish (Matsunami) precoated with poly-d -lysine (Sigma) and incubated at 37 °C in 5% CO2. Images in the differential interference contrast (DIC) and GFP channels were captured with an LCV110 microscope (Olympus) every 20 min for 96 h. Semiautomated cell tracking was done by Fiji software with the Trackmate plugin (65 (link)). Cells, which had already expressed d2EGFP at the beginning of the observation, were excluded from analysis, because the starting point for expression was unknown. To analyze single-cell dynamics of d2EGFP expression, normalized fluorescence intensities are plotted against time. The starting time (t = 0) is the time at which the cell started expressing d2EGFP above background level.
4
Time-lapse Imaging of Differentiation
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Rcho-1 cells were cultured in a 6 cm dish (Matsunami, Japan) and pEF1a/Gal-4 or an empty control vector was transfected as described above. Time-lapse recording began after exchanging the medium from RPMI1640 to differentiation medium (NCTC/1% horse serum) at 48 hrs post transfection using a LCV110 microscope (Olympus, Japan) which acquired a z-stack image every 30 minutes, for a total of 12 hrs. All file handling and image analysis was done in ImageJ (http://imagej.nih.gov ). The number of adherent ZsGreen-positive cells was counted 12 hrs after time-lapse recording was started. The proportion per unit time of adherent cells was represented as p. By use of binomial distribution based on the 4-th trial number and p, the proportion of cells in contact with 0–3 ZsGreen-positive cells was hypothesized to be ((1-p)3, 3(1-p)2p, 3(1-p)p2, p3) which was derived from the following steps; Step1(1, 0, 0, 0), Step2(1-p, p, 0, 0), Step3((1-p)2, 2(1-p)p, p2, 0), Step4((1-p)3, 3(1-p)2p, 3(1-p)p2, p3). Each p of Gal-4 or empty vector was calculated using a least squares method by comparing the experimental data and the components of Step4.
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