Timelapse imaging was performed using a Nikon Ti-U inverted microscope equipped with a stage top incubator maintained at 37°C in a 5% CO2, 90% relative humidity atmosphere (Pathology Devices, Inc.). Images were acquired every 30 min using a Plan Fluor 10×/0.3 NA air objective (Nikon) and a Hamamatsu Orca-100 camera. Individual GFP-labeled cells were tracked from the aligned image sequences using the “Manual Tracking” plugin in ImageJ.
Orca 100 camera
Manufactured by Hamamatsu Photonics
Sourced in United States, Netherlands
The ORCA-100 is a high-performance scientific camera developed by Hamamatsu Photonics. It features a 1.3-megapixel scientific CCD sensor with high quantum efficiency and low noise characteristics. The camera is designed for a wide range of scientific and industrial applications requiring accurate and sensitive image capture.
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Lab products found in correlation
Ti u inverted microscope, by Nikon (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Metamorph acquisition software, by Molecular Devices (1 mentions)
C3100mp, by Thermo Fisher Scientific (1 mentions)
Ethd 1, by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
5 protocols using orca 100 camera
1
Time-lapse Imaging of GFP-labeled Cells
2
Asbestos-Induced DNA Damage in Mesothelial Cells
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Human pleural mesothelial cells were exposed to LGM2605 (50 µM) 4 h prior to asbestos challenge (20 µg/cm2), and cells were evaluated at 0, 2, and 6 h post asbestos exposure. Mesothelial cells were fixed, permeabilized, and immunostained using anti-γH2AX (1:200) as primary antibody followed by secondary antibody conjugated to Alexa488 (green). Fluorescence imaging was performed at λ ex 488 nm on a Nikon TMD fluorescence microscope equipped with a Hamamatsu ORCA-100 camera. All fluorescent cell images were acquired at the same magnification (10× lens), exposure (250 ms exposure), and acquisition settings using the MetaMorph software (Version 7.7, Molecular Devices, Downingtown, PA, USA). The fluorescent images of cells were processed and quantitated the use of ImageJ software (Fiji Version, National Institutes of Health, Bethesda, MD, USA). For each experimental condition, 3–4 fields were imaged for n = 3 independent experiments. The intensity of cells in each field was integrated and normalized to area to obtain the total fluorescence intensity, and expressed as arbitrary fluorescence intensity units (AFU). Scale bar = 15 μM.
3
Intracellular ROS Levels in Macrophages
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Levels of intracellular ROS were determined using CellROX® Green Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Elicited macrophages from WT and Nrf2−/− mice were exposed to LGM2605 (50 µM and 100 µM) 4 h prior to asbestos challenge (20 µg/cm2) and cells were harvested at 24 h post asbestos exposure. Asbestos-treated and untreated cells were incubated with 5 μM CellROX® Green Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37 °C after which cells were washed with phenol red free RPMI and imaged on a Nikon TMD fluorescence microscope (Nikon Diaphot TMD, Melville, NY, USA) equipped with a Hamamatsu ORCA-100 camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). All fluorescent cell images were acquired at the same exposure and offset settings using the MetaMorph acquisition software (Version 7.7, Molecular Devices, Downington, PA, USA). The fluorescent images of cells were processed and quantitated for CellROX® Green Reagent fluorescence by the use of ImageJ software (Fiji Version, National Institutes of Health, Bethesda, MD, USA). The intensity of cells in each field was integrated to obtain the total fluorescence intensity of a particular field. Scale bar = 20 μm.
4
Laser-based Cell Viability Assay
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Cell death was determined by postlaser exposure in the same manner for all sample types. After exposures, the exposure buffer was replaced with complete medium and cells were incubated at 37°C and 5% for at least 1 h to allow recovery or damage progression. After recovery, cells were stained for 15 min at 37°C for viability using calcein-AM (C3100MP, Life Technologies, Grand Island, New York) and ethidium homodimer 1 (EthD1, E3599, Life Technologies) in complete HBSS. Laser-damaged sites were identified by imaging on an Olympus CK-40 using EthD1 channel (bandpass exciter of 475–545 nm and a barrier filter at 590 nm) and Calcein-AM channel (bandpass exciter of 460–490 nm and a bandpass emitter of 490–530 nm). Monochrome images were taken using a Hamamatsu ORCA 100 camera.
Damage threshold irradiance values ( ) were determined using the Probit54 ,55 method, as previously described.8 (link)– 10 (link, link) The Probit output included 95% confidence intervals [fiducial limits (FLs)] related to the value. Probit slope values were determined by taking the first derivative of the Probit probability curve at the 50% value.
Damage threshold irradiance values ( ) were determined using the Probit54 ,55 method, as previously described.8 (link)
5
Nucleolus Morphology Analysis in Yeast
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A co-localization study was performed using exponentially growing KAY575 cells transformed with pWJ1322 on SC-URA media. Cells were examined using an Axio Imager M2 fluorescence microscope (Zeiss) equipped with 38HE and 20HE filter sets for Las17-GFP and Nop1-DsRed, respectively and was documented using Axio Vision 4.8.
For the nucleolus morphology assay, RLY1 (wild type, WT) or RLY157 (las17Δ) cells carrying pWJ1322 (Nop1-DsRed) were grown on SC-URA media, washed twice in PBS and viewed with an Eclipse (Nikon, Amsterdam, Netherlands) fluorescence microscope equipped with a Hamamatsu ORCA 100 camera. Images were collected using Lucia G software. The morphology of the nucleolus was analyzed in at least 350 cells.
To analyze nucleolus morphology during division, yeast transformants grown on selective media (SC-ura) were cultivated in YPD to the exponential growth phase, and then 7.5 μg/ml was added to the WT and las17Δ strains. YPD is superior to synthetic media for nocodazole synchronization. After further cultivation for 2 hours, cells were washed and then suspended in fresh medium. At 30, 60, 90 and 120 min after the removal of nocodazole, cells were observed, and images were collected as described above.
For the nucleolus morphology assay, RLY1 (wild type, WT) or RLY157 (las17Δ) cells carrying pWJ1322 (Nop1-DsRed) were grown on SC-URA media, washed twice in PBS and viewed with an Eclipse (Nikon, Amsterdam, Netherlands) fluorescence microscope equipped with a Hamamatsu ORCA 100 camera. Images were collected using Lucia G software. The morphology of the nucleolus was analyzed in at least 350 cells.
To analyze nucleolus morphology during division, yeast transformants grown on selective media (SC-ura) were cultivated in YPD to the exponential growth phase, and then 7.5 μg/ml was added to the WT and las17Δ strains. YPD is superior to synthetic media for nocodazole synchronization. After further cultivation for 2 hours, cells were washed and then suspended in fresh medium. At 30, 60, 90 and 120 min after the removal of nocodazole, cells were observed, and images were collected as described above.
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