Cells were grown on coverslips and incubated with 10 μM BrdU for 48 h before HU treatment at 2 mM for 4 h. Cells were permeabilized with 0.3% Triton X-100 in PBS for 3 min at 4 °C, washed three times in PBS, and fixed with 4% paraformaldehyde for 10 min at 4 °C. After three PBS washes, cells were blocked with 5% BSA, then incubated with mouse anti-BrdU (BU-1, 1:300) antibody in 1% BSA for 2 h. After three PBS washes, cells were incubated with 1:1000 Alexa Fluor 488 goat anti-mouse IgG for 45 min in 1% BSA. After three PBS washes, coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Lab) and analyzed with the Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera. Corrected total cell fluorescence (CTCF) intensity was quantified and calculated using Fiji and analyzed with Prism (GraphPad).
Eclipse ts2r fl inverted fluorescence microscope
Manufactured by Nikon
The Eclipse Ts2R-FL is an inverted fluorescence microscope manufactured by Nikon. It is designed for high-quality imaging of fluorescent samples. The microscope features a compact and ergonomic design, and it is equipped with a variety of illumination options, including LED and mercury lamp illumination.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium containing dapi, by Vector Laboratories (2 mentions)
Nis elements research br software, by Nikon (2 mentions)
Ds qi2 digital camera, by Nikon (2 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Ds qi2, by Nikon (1 mentions)
Mouse anti biotin, by Jackson ImmunoResearch (1 mentions)
Mouse anti brdu antibody, by BD (1 mentions)
Dapi containing mounting medium, by Vector Laboratories (1 mentions)
Sybr gold staining solution, by Thermo Fisher Scientific (1 mentions)
Comet assay kit, by Bio-Techne (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
6 protocols using eclipse ts2r fl inverted fluorescence microscope
1
Quantifying BrdU Incorporation in Cells
2
Immunofluorescence Staining of Cultured Cells
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Cells were seeded on coverslips at least 24 h before any drug treatment. To stop the treatment, cells were washed with cold PBS on ice, then fixed with 4% paraformaldehyde for 10 min at 4 °C. After three washes with PBS, cells were permeabilized with 0.3% Triton X-100 in PBS for 3 min on ice. Following permeabilization, cells were washed three times with PBS, and blocked for 45 min at RT using 5% BSA in PBS. Subsequently, cells were incubated with primary antibodies diluted in 1% BSA for 1 h at RT. After three PBS washes, cells were incubated with secondary antibodies coupled to fluorochromes diluted in 1% BSA for 45 min at RT. After three additional PBS washes, coverslips were mounted onto microscope slides using Vectashield mounting medium containing DAPI (Vector Lab). Coverslips were analyzed with an Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera. Fluorescence images were analyzed using Fiji, and quantification data were processed by Prism (GraphPad).
3
Quantification of Nascent DNA and MCM6 Interaction
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Cells were grown on poly-l -lysine coated glass coverslips and treated for 8h with the indicated drugs. Twelve minutes before the end of treatment, EdU was added to the media at a final concentration of 125 μM. Cells were fixed with 4% PFA for 10 min at RT, washed in PBS and stored overnight at 4 °C in fresh PBS and wrapped in foil. Cells were then permeabilized with 0.3% Triton X-100 in PBS for 3 min on ice. Following PBS washes, a Click-iT reaction was performed to tag the EdU alkyne using 10 μM biotin-azide, 2 mM CuSO4 and 10 mM sodium ascorbate for 1 h at RT in a humidified chamber protected from light. Cells were then washed in PBS once before proceeding with the PLA assay between biotin-labeled nascent DNA and MCM6 following the manufacturer's instructions (Duolink, Millipore Sigma). Rabbit anti-biotin antibody (Bethyl Laboratories, #A150-109A; 1:3000) was used together with either mouse anti-MCM6 (Santa Cruz, sc-393618, 1:500) or mouse anti-biotin (Jackson ImmunoResearch, #200-002-211, 1:2000) to control for EdU uptake. Coverslips were mounted on slides and imaged with an Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with a Nikon DSQi2 camera. Images were analyzed using the NIS-Elements Research BR software (Nikon) and quantifications were analyzed using Prism (GraphPad).
4
Quantifying BrdU Incorporation in U2OS Cells
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U2OS Flp-In cells were reverse transfected with siRNA oligos in 6-cm dishes, induced with doxycycline (100 ng/mL) and seeded in 12-well plates on glass coverslips 48 h after transfection. The following day, cells were pulse-labelled with 10 μM BrdU for 25 min in pre-equilibrated media, washed once with warm PBS and treated or not with 4 mM HU for 2 h. After a wash with cold PBS on ice, cells were permeabilized with PBS/0.5% Triton X-100 for 5 min on ice, washed again with PBS three times, and fixed with 4% paraformaldehyde for 10 min at RT. After three PBS washes, cells were blocked with 5% BSA for 1 h at RT, then incubated with mouse anti-BrdU antibody (BD, #347580, 1:10) in 1% BSA for 1 h at RT. After three PBS washes, cells were incubated with goat anti-mouse Alexa Fluor 488 IgG at 1:1,000 in 1% BSA for 45 min at RT. After three PBS washes, coverslips were mounted using DAPI-containing mounting medium (Vector Lab) and analyzed with the Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera. Cells with more than 10 foci were counted as positive. Percentages of positive cells were quantified and analyzed by Prism (GraphPad).
5
DNA Comet Assay for Genotoxicity
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DNA comet assay was performed using the CometAssay kit (4250-050-K, Trevigen) according to the manufacturer's protocol. Briefly, 25 μl of the sample cell suspension at 2 × 105 cells/ml were combined with 225 μl of low-melting agarose (LMAgarose) (1:10 ratio, v/v) and 50 μl of this mixture was quickly spread onto the provided comet slides (Trevigen). Following solidification at 4°C, slides were immersed in cold lysis solution for 1 h at 4°C then placed in freshly prepared alkaline unwinding solution (20 mM NaOH, 1 mM EDTA) for 20 min at RT. Electrophoresis of unwound DNA was performed at 21 V for 30 min. Slides were then washed twice with dH2O for 5 min, dehydrated with 70% ethanol for 5 min, dried, and stained with SYBR Gold staining solution (Thermo Fisher) for 30 min. Comets were imaged at 10x magnification with an Eclipse Ts2R-FL inverted fluorescence microscope (Nikon) equipped with a Nikon DSQi2 digital camera and analyzed using OpenComet on Fiji and Prism (GraphPad). Up to at least 100 individual nuclei were evaluated per group.
6
Immunofluorescence Staining of Adherent Cells
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Coverslips were coated with poly-l -lysine (Millipore Sigma) for 15 min, and cells were seeded at least 24 h before any drug treatments. Following treatments, cells were washed once with cold PBS on ice and fixed with 4% paraformaldehyde for 10 min at RT. After three washes with PBS, cells were permeabilized with 0.3% Triton X-100 in PBS for 3 min on ice. Following permeabilization, cells were washed three times with cold PBS and blocked for 1 h at RT using 5% BSA in PBS. Subsequently, cells were incubated with primary antibodies diluted in 1% BSA for 2 h at RT. After three PBS washes, cells were incubated with secondary antibodies coupled to fluorophores diluted in 1% BSA for 45 min at RT. Following three additional washes with PBS, coverslips were mounted onto glass microscope slides using Vectashield mounting medium containing DAPI (Vector Lab). Coverslips were imaged with an Eclipse Ts2R-FL inverted fluorescence microscope (Nikon) equipped with a Nikon DSQi2 digital camera and analyzed using NIS-Elements, Research BR software (Nikon). Quantification data was processed using Prism (GraphPad).
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