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.
Ds qi2 digital camera
Manufactured by Nikon
Sourced in Japan, United States
The DS-Qi2 is a digital camera designed for microscopy applications. It features a high-resolution sensor and advanced image processing capabilities to capture detailed micrographs. The camera is compatible with a variety of microscope systems and can be used for a range of scientific and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ts2r fl inverted fluorescence microscope, by Nikon (2 mentions)
Vectashield dapi, by Vector Laboratories (2 mentions)
Eclipse ts2r microscope, by Nikon (2 mentions)
Nis element, by Nikon (2 mentions)
Sybr gold staining solution, by Thermo Fisher Scientific (1 mentions)
Comet assay kit, by Bio-Techne (1 mentions)
Nis elements software, by Nikon (1 mentions)
Rabbit anti rad51, by Abcam (1 mentions)
Vectashield mounting medium containing dapi, by Vector Laboratories (1 mentions)
Nis elements research br software, by Nikon (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Eclipse ti u fluorescent microscope, by Nikon (1 mentions)
Nis elements image analysis software, by Nikon (1 mentions)
Eclipse 80i fluorescence microscope, by Nikon (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AppliChem (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
9 protocols using ds qi2 digital camera
1
DNA Comet Assay for Genotoxicity
2
Integrin Trafficking Visualization
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Cells were grown on coverslips and serum starved at 37 °C overnight. The cells were transferred to ice to cool down. Surface integrin β4 were labeled with anti-Alexa 488-CD104 antibody (Invitrogen, #MA5-23641) in serum-free medium containing 0.01% bovine serum albumin (BSA) for 1h at 4 °C. Labeled cells were washed two times with cold serum-free medium containing 0.01% BSA and subsequently transferred to prewarmed free-serum medium. Integrin internalization was allowed at 37 °C for 2 h. For recycling, internalized integrin was stimulated with EGF for different times at 37 °C. The cells were washed twice with PBS, fixed in paraformaldehyde (PFA) and mounted with Fluoromount-G (Southern Biothech, Birmingham, AL, USA) or Vectashield DAPI (Vector lab, Burlingame, CA, USA) for. Integrin trafficking was monitored by immunofluorescence microscope. Protein localization was captured at 60× oil magnification and DIC was captured at CFI Plan Apo Lambda 60× Oil magnification using a Nikcon Eclipse Ts2R microscope with Nikon DSQi2 Digital Camera. All images were analyzed using NIS-Elements software (NIS-Elements advanced research 4.5 version, Nikon, Tokyo, Japan) and processed using Adobe Photoshop software (Adobe Photoshop CC 2015).
3
Immunofluorescence analysis of DNA damage response
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Cells placed on cover slips were exposed to Olaparib, CMM489, or the combination treatment. After 3 days, the cells were washed in PBS, fixed in 4% formaldehyde, permeabilized in 0.2% Triton X-100, and blocked in 1% bovine serum albumin (BSA) in PBST. The cells were then incubated with primary antibodies (rabbit anti-RAD51; Abcam, Cat. No. ab133534, mouse anti-γHA2X; Abcam, Cat. No. ab26350) for 2 h at room temperature or overnight at 4 °C and incubated with appropriate fluorophore-conjugated secondary antibodies for 1 h at room temperature. Slides were mounted with Vectashield DAPI (Vector lab). Immunofluorescence was visualized using a Nikon Eclipse Ts2R microscope with a Nikon DSQi2 Digital Camera. All images were analyzed using NIS-Elements software (NIS-Elements advanced research 4.5 version, Nikon, Tokyo, Japan).
4
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).
5
Crataegus Extract Effects on Cell Morphology
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Cells were seeded at a density of 3 × 105 cells per 6-cm culture dish (Sarstedt, Nümbrecht, Germany). After 18 h, cells were treated with Crataegus extracts (250 µg/mL) for 48 h. Non-treated cells were used as a control. Cells incubated with DMSO at concentrations of 0.02–1.5% dependent on the flower extract concentrations (see Figure S1 ) served as an additional control for the flower extract analysis. Cell morphology was analyzed by light microscopy (Nikon Eclipse Ti-U fluorescent microscope equipped with a 20× objective and DS-Qi2 digital camera, Tokyo, Japan).
6
Fluorescence Microscopy of Induced Cells
7
Immunostaining of MDA-MB-231 Cells
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MDA-MB-231 cells were plated on 50 μg/mL rat tail type I collagen-coated PAGs or 12 mm coverslips. The cells were fixed with 3.7% paraformaldehyde (Sigma-Aldrich) for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. To block background signals, the samples were incubated with 2% bovine serum albumin (BSA) in 0.1% Triton X-100 in PBS for 1 h. The cells were incubated with primary antibodies for 1 h and then with fluorescein-conjugated secondary antibodies for 1 h. The samples were mounted on glass slides with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA, #0100-01) and observed under an Eclipse 80i fluorescence microscope (Nikon) equipped with a DS-Qi2 digital camera (Nikon). Captured images were processed using the NIS-Elements image analysis software (Nikon).
8
Fluorescence Microscopy Protocol
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For fluorescence microscopy,
cells were passaged onto 6-well plates and induced for 48 h prior
to imaging. Images were acquired on a Nikon TiE inverted fluorescence
microscope equipped with a Nikon DSQi2 Digital Camera using CFI Plan
Fluor 10×/0.30/16.00 objective and ET Sputter Coat Ex470/40 Dm495
Bar525/50 FITC/GFP filter for EGFP and a ET Sputter Coat Ex560/40
Dm585 Bar630/75 TX Red filter for mCherry. Composite images with scale
bars were assembled in Nikon NIS Elements.
cells were passaged onto 6-well plates and induced for 48 h prior
to imaging. Images were acquired on a Nikon TiE inverted fluorescence
microscope equipped with a Nikon DSQi2 Digital Camera using CFI Plan
Fluor 10×/0.30/16.00 objective and ET Sputter Coat Ex470/40 Dm495
Bar525/50 FITC/GFP filter for EGFP and a ET Sputter Coat Ex560/40
Dm585 Bar630/75 TX Red filter for mCherry. Composite images with scale
bars were assembled in Nikon NIS Elements.
9
Immunostaining of Drosophila Polytene Chromosomes
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Drosophila 3 rd instar larvae were cultured at 18°C under standard conditions. Polytene chromosome staining was performed as described (43) . The following primary antibodies were (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 11, 2020. ; https://doi.org/10.1101/2020.11.11.378323 doi: bioRxiv preprint used: rabbit anti-MSl1 at 1:100 dilution, mouse anti-FLAG at 1:100 dilution. The secondary antibodies were Alexa Fluor 488 goat anti-rabbit 1:2,000 and Alexa Fluor 555 goat anti-mouse 1:2,000 (Invitrogen). The polytene chromosomes were co-stained with DAPI (AppliChem).
Images were acquired on the Nikon Elclipse Ti fluorescent microscope using Nikon DS-Qi2 digital camera, processed with ImageJ 1.50c4 and Fiji bundle 2.0.0-rc-46. 3-4 independent staining and 4-5 samples of polytene chromosomes were performed with each MSL1-expressing transgenic line.
The copyright holder for this preprint this version posted November 11, 2020. ; https://doi.org/10.1101/2020.11.11.378323 doi: bioRxiv preprint used: rabbit anti-MSl1 at 1:100 dilution, mouse anti-FLAG at 1:100 dilution. The secondary antibodies were Alexa Fluor 488 goat anti-rabbit 1:2,000 and Alexa Fluor 555 goat anti-mouse 1:2,000 (Invitrogen). The polytene chromosomes were co-stained with DAPI (AppliChem).
Images were acquired on the Nikon Elclipse Ti fluorescent microscope using Nikon DS-Qi2 digital camera, processed with ImageJ 1.50c4 and Fiji bundle 2.0.0-rc-46. 3-4 independent staining and 4-5 samples of polytene chromosomes were performed with each MSL1-expressing transgenic line.
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