Mitotic cells were collected by mitotic shake-off and swollen in hypotonic buffer (0.03 M sodium acetate) at 37ºC for 25 min, then fixed in a freshly prepared 3:1 mix of methanol:glacial acetic acid. Nuclear preparations were dropped onto slides pre-soaked in 45% acetic acid and stained with Giemsa. Metaphases were viewed with a Leica DMI6000B inverted microscope equipped with a HCX PL APO 100x/1.4-0.7 oil objective. Images were acquired using a Leica DFC350 FX R2 digital camera and LAS-AF software (Leica). Brightness levels and contrast adjustments were applied using Photoshop CS3 (Adobe). 60 metaphases from two independent experiments were quantified per condition.
Dfc350fxr2
Manufactured by Leica
Sourced in Germany
The DFC350FXR2 is a high-performance digital camera for microscopy applications. It features a 3.2-megapixel CMOS sensor and is capable of capturing images with a resolution up to 2048 x 1536 pixels. The camera is designed for use with Leica microscopes and offers a selection of exposure times and gain settings to accommodate a variety of imaging scenarios.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (3 mentions)
Hcx pl apo 100x 1.4 0 7 oil objective, by Leica (2 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Dmi6000b, by Leica (1 mentions)
Mlc400, by Agilent Technologies (1 mentions)
Csu x1, by Nikon (1 mentions)
Ti e platform, by Agilent Technologies (1 mentions)
Af6000 lx, by Leica (1 mentions)
Dm5000b, by Leica (1 mentions)
Af6000 lx system, by Leica (1 mentions)
Dm5000b microscope, by Leica (1 mentions)
8 protocols using dfc350fxr2
1
Mitotic Cell Chromosome Imaging
2
Mitotic Cell Chromosome Imaging
3
Live/Dead Cell Imaging Assay Protocol
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Cells were seeded into 8-well chamber slides (Thermo Scientific, Rochester, NY, USA) at a concentration of 100,000 cells/400 µL/well in full medium, allowed to attach overnight, followed by serum-starvation (1% FBS) for another 24 hours and incubated for 24 and 48 hours with the test substances that were diluted in fresh serum-starved medium. The fluorescent LIVE/DEAD Cell Imaging Assay (488/570 nm; Lifetechnologies, Eugene, OR, USA) was performed following the manufacturer's instructions.68 –71 (link) Cells were visualized under 100x magnification using a fluorescent microscope (Leica DMI 6000B, Wetzlar, Germany) with a monochrome digital camera (DFC350FXR2; Leica) and the filter sets Cy5/Y3 (exposure time 1030 ms, intensity 3, and gain 6.1) and Alexa488/L5 (exposure time 545 ms, intensity 3, and gain 6.1). Negative controls were performed by microscoping chamber slides without addition of fluorescent solution.
Three digital images per well were randomly taken (Leica Application Suite Advanced Fluorescence software 2.7.0.9329), blinded, and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).72 (link) Cell survival was calculated by building a quotient out of the area of living cells and the total cell area for each image.
Three digital images per well were randomly taken (Leica Application Suite Advanced Fluorescence software 2.7.0.9329), blinded, and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).72 (link) Cell survival was calculated by building a quotient out of the area of living cells and the total cell area for each image.
4
Live-cell Imaging of HeLa Cells
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HeLa cells were analyzed 48 h after transfection using an inverted Leica AF 6000 LX TIRF microscope equipped with a 100× objective (HCX PL APO 100× 1.47 Oil; Leica) and a 12-bit CCD camera (DFC350FXR2; Leica); the temperature was 28–30°C. The penetration depth of the evanescent wave was 70 and 90 nm, respectively, for the lasers exciting EGFP and mCherry. The exposure time was set individually for each cell and channel to obtain the best signal-to-noise ratio. Image sequences were acquired every 0.5–5 s for ∼10 min with LAS AF software (Leica Microsystems, Wetzlar, Germany), and data were postprocessed using NIS Elements AR 4 software.
5
Spinning Disc Confocal and VAEM Imaging
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Image series (videos) were recorded for 2 min unless stated otherwise from seedlings mounted in water on chambered slides as described previously [3 (link), 25 (link), 26 (link)]. SDCM recordings were performed using an inverted spinning disc confocal microscope (Yokogawa CSU-X1 on a Nikon Ti-E platform, laser box Agilent MLC400, camera Andor Ixon) with plan apochromat × 100 oil (NA = 1.45) lens, laser lines set at 488 and 561 nm, and image interval 1 s. VAEM recordings were generated using the Leica AF6000 LX fluorescence platform with integrated TIRF module and a Leica DFC350FXR2 digital camera, with plan apochromat × 100 oil (NA = 1.46) lens, 400 nm peak excitation, and 210 ms exposure time, allowing for image interval 0.5 s.
6
Rotifer Ecotoxicology Assay Protocol
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Ten juveniles were placed in each well in 6 multi-well plates, in a volume of 5 mL of standard freshwater medium per well (Table S1). Modelled concentrations as EC 10 (effect concentration reducing 10 % of population growth rate) were tested against a negative control with clean freshwater medium (6 replicates) for a period of 48 h. After this period rotifers were frozen with liquid nitrogen and stored in -80 • C.
The rotifers from this 48 h sub-lethal test were prepared with the following fluorescence markers: 5 μM CM-H 2 DCFDA, 15 μM propidium iodide and 100 μL of the anti-fading reagent containing 4 ′ ,6-diamidino-2-phenylindole (DAPI). Rotifers were unfrozen, centrifuged (10,000 g for 5 min) to remove the excess of medium. Then, rotifers were suspended in freshwater medium and incubated with CM-H 2 DCFDA and propidium iodide for 15 min in the dark. After this period, the dye was removed by centrifugation (10,000 g; 5 min), and clean freshwater medium was added to resuspend rotifers. Finally, the anti-fading reagent was added before loading the samples onto the microscope slides. Each slide was scanned through an epifluorescence microscopy (160× and 200×, Leica DM5000B), and images were acquired with a digital camera (Leica DFC 350 FX R2) using the software LAS AF (Ver. 1.4.1).
The rotifers from this 48 h sub-lethal test were prepared with the following fluorescence markers: 5 μM CM-H 2 DCFDA, 15 μM propidium iodide and 100 μL of the anti-fading reagent containing 4 ′ ,6-diamidino-2-phenylindole (DAPI). Rotifers were unfrozen, centrifuged (10,000 g for 5 min) to remove the excess of medium. Then, rotifers were suspended in freshwater medium and incubated with CM-H 2 DCFDA and propidium iodide for 15 min in the dark. After this period, the dye was removed by centrifugation (10,000 g; 5 min), and clean freshwater medium was added to resuspend rotifers. Finally, the anti-fading reagent was added before loading the samples onto the microscope slides. Each slide was scanned through an epifluorescence microscopy (160× and 200×, Leica DM5000B), and images were acquired with a digital camera (Leica DFC 350 FX R2) using the software LAS AF (Ver. 1.4.1).
7
Fluorescence Imaging Protocol for Cell Analysis
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Fluorescence images were taken using a Leica Microsystems AF 6000 LX system equipped with a Leica camera DFC350FXR2. Imaging was done at a resolution of 8 bits and binning 1 × 1. A Leica CY3 filter was used for excitation and emission with a dry 5× PLAN objective. The exposure time was kept at 25 s, the gain at 10.0 and intensity at 5 a.u. The mean fluorescence intensity was evaluated from 33,792 pixels in a rectangular region of interest (ROI). The ImageJ software was used for image analysis.
8
Yeast Cell Fluorescent Microscopy
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Yeast cells were grown, as described above, and visualized by fluorescent microscopy. A volume of 1 mL of growing yeast cells was collected and concentrated by a factor of 10. 5 μL of each sample was then directly visualized, without fixation, on a Leica DM5000B microscope with appropriate filters. The resulting images were acquired with a Leica DFC 350FX R2 digital camera using the LAS AF software.
Images were then processed in the Adobe Photoshop CC 2018 (Adobe Systems).
Images were then processed in the Adobe Photoshop CC 2018 (Adobe Systems).
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