These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair. Abstract Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage.
In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols lidase és prostatitis the field of DNA repair, Fluorescence Immunostaining IF and Chromatin Immunoprecipitation ChIPwhich in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and lidase és prostatitis fashion, respectively.
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These lidase és prostatitis provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair. Introduction Log in or Start trial to access full content. These assaults can derive from environmental sources, such as UV light or irradiation, as well as from endogenous sources, such as metabolic by-products caused by oxidative stress or replication errors1,2.
These lesions can affect the integrity of either one or both DNA strands, and if the generated errors become persistent, it frequently leads to translocations and genome instability, which may result in tumorigenesis3,4.
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
To maintain genome integrity, multiple repair systems have been developed during evolution. According to the chemical and physical properties of specific types of DNA damage, multiple repair mechanisms can be activated.
Mismatches, abasic sites, single-strand breaks, and 8-oxoguanine 8-oxoG can be removed either by mismatch repair or base-excision repair pathway5,6.
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Regarding the cell cycle phase, following DNA double-strand break induction, two sub-pathways can be activated: non-homologous end joining NHEJ and homologous recombination HR 1,9.
NHEJ, which is the dominant pathway in resting cells, can be activated lidase és prostatitis all cell cycle phases, representing a faster but error-prone lidase és prostatitis On the other hand, HR is an error-free pathway, in which the DSBs are repaired based on sequence-homology search of the sister chromatids, therefore it is mainly present in S and G2 cell cycle phases Therefore, MMEJ is considered to be error-prone and highly mutagenic The DDR is activated as a response to the recruitment and extensive spreading of initiator key players of the repair process around the zab prosztatitis, contributing to the formation of a repair focus.
This early event is responsible for the recruitment of additional repair factors and the initiation of downstream repair processes.
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Although the exact function of the recruited proteins at the repair focus has not yet been fully characterized, the formation and lidase és prostatitis dynamics of repair foci have been investigated by several laboratories. These markers are extensively used to follow the repair kinetics, but their precise role during the repair process remains elusive.
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Due to the great importance yet poor understanding of DNA repair-related cellular processes, several methods have been developed so far to induce and visualize the DDR. Various methods and systems have been established to induce the desired type of DNA damage. Here, we focus on the endonuclease-based techniques currently used to study the DDR in mammalian and yeast cells.
Aside from highlighting the principles of these techniques, we emphasize both their advantages and disadvantages. Subscription Required. Please recommend JoVE to your librarian. Protocol Log in or Start trial to access full content. Aspirate the medium and wash the cells with 1x PBS. When the cells detach, stop the trypsin activity by adding culture medium to the cells, yielding a cell suspension.
Lidase és prostatitis the cells using a cell counting chamber.
Incubate the cells with the NCS-containing medium for 15 min, then wash them with prostatitis gyógynövények PBS and add fresh, supplemented culture medium to the cells.
Otherwise, use appropriate agent i. Each incubation and washing step except the antibody incubation should be performed on an orbital shaker with gentle agitation.
Following DSB induction and incubation of cells, remove the medium from the attached cells and wash the cells once with 1x PBS. Permeabilization of cells Remove the fixing solution and wash cells three times with 1x PBS for 5 min each. Remove the PBS and add 0. Incubate the samples for 20 min.
Blocking of non-specific binding sites Wash the cells three times with 1x PBS. Immunofluorescence staining Add the proper amount of primary antibody i.
Lidase és prostatitis each coverslip upside down onto a paraffin film over a 10 µL droplet of the diluted anti-γH2AX antibody. Incubate the samples in a humidity chamber for 1. Place the coverslips back being side up into the well plate and wash three times for 5 min with 1x PBS. Place each coverslip upside down onto a paraffin film over a 10 µL droplet receptek a prosztata gyulladására the diluted antibody.
Incubate the samples in a humidity chamber at 4 °C for 1 h. Before removing the last PBS washing solution, gently take out the coverslips using a tweezer and needle and then place them upside down onto glass slides with droplets of mounting medium supplemented with DAPI.
When the mounting medium dries, it is recommended to seal the edges of the coverslips with nail polish to prevent shriveling of the samples.
- Zsófia Sztupinszki - Google Tudós
- List of Posters – DDRS
- Élelmiszer krónikus prosztatával
- - Я - добавил не должно всем, но беспочвенными фантазиями.
Remove the culture medium and wash the cells twice with ice-cold 1x PBS. NOTE: Formaldehyde is volatile; always prepare a fresh working solution.
In some cases, the formaldehyde solution contains methanol to stabilize it, but it is better to use a methanol-free solution to avoid interference with downstream reactions.
Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas
Stop the fixation with mM glycine and incubate on an orbital shaker with gentle agitation for 5 min at 25 °C. Place the plates on ice and wash twice with ice-cold 1x PBS. Scrape the cells in ice-cold 1x PBS and transfer them into 15 mL conical tubes.
Centrifuge the cells at 2, x g for 5 min at 4 °C. Centrifuge the cell suspension at 2, x g for 5 min at 4 °C.
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- Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas
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Carefully discard the supernatant and resuspend the pellet inµL of nuclear lysis buffer 50 mM Tris-HCl pH 8. Transfer the lysate into a polystyrene conical tube suitable for sonication. The solution should turn transparent following sonication.
Sonicate the lysate to shear DNA to an average fragment size ofbp. NOTE: The appropriate sonication cycles and conditions should be set according to the cell type and the sonication equipment.
Reversal of crosslinking, determination of sonicated fragment sizes Take out µL of the sonicated sample to verify the fragment size of the sonicated chromatin. Add 0. Incubate the samples at 65 °C overnight.
Vortex for 1 min. Centrifuge at 13, x g for 10 min.
Transfer the upper aqueous phase to a new microcentrifuge tube. Add 1 volume chloroform-isoamyl alcohol mix to each sample. Add 2. Centrifuge the samples at 13, x g for 10 min at 4 °C.
Remove the ethanol and air dry the pellet. Resuspend the pellet in 10 µL of TE. Run the samples on a 0. The sonicated chromatin size should be around bp.
Pituitary adenoma xenografts were generated in immunocompromised mice.