Quantifying DNA Damage Foci Post-Irradiation

Cells were seeded on cover slips at least 2 days prior to irradiation and were fixed in 70% EtOH for 10 min at the indicated time points after exposure. Cells were permeabilised in 0.2% Triton-X in phosphate-buffered saline (PBS) and incubated with anti-phospho-Histone H2AX antibody Ser139 (Upstate/Millipore) followed by secondary anti-mouse IgG fluorescein isothiocyanate (FITC) (Sigma-Aldrich), both in PBS containing 2% bovine serum albumin (BSA). Coverslips were counterstained using DAPI and mounted on an objective glass in Vectashield. Photos were taken, and the total number of γH2AX foci per cell was scored in 50 cells per treatment and experiment using a macro for ImageJ version 1.43u, as previously described [30 (link)].

Free full text: Click here

Svetličič M., Bomhard A., Sterr C., Brückner F., Płódowska M., Lisowska H, & Lundholm L. (2020). Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells. Cells, 9(5), 1165.

Publication 2020

anti-mouse IgG fluorescein isothiocyanate (FITC)

Anti antibody Anti igg Bovine serum albumin Cells Dapi Etoh Fluorescein Histone h2ax Irradiation Isothiocyanate Mouse Phosphate Saline

Corresponding Organization : Stockholm University

Other organizations : Jan Kochanowski University

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Irradiation

dependent variables

Total number of γH2AX foci per cell

control variables

Cells were seeded on cover slips at least 2 days prior to irradiation
Cells were fixed in 70% EtOH for 10 min at the indicated time points after exposure
Cells were permeabilised in 0.2% Triton-X in phosphate-buffered saline (PBS)
Cells were incubated with anti-phospho-Histone H2AX antibody Ser139 (Upstate/Millipore) followed by secondary anti-mouse IgG fluorescein isothiocyanate (FITC) (Sigma-Aldrich), both in PBS containing 2% bovine serum albumin (BSA)
Coverslips were counterstained using DAPI and mounted on an objective glass in Vectashield

controls

No positive or negative controls were explicitly mentioned in the input protocol.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!