Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

Summary

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Abstract

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely γH2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of γH2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of γH2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of γH2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

Introduction

DNA damage is continuously induced by agents that can be endogenous, such as ROS generated by cellular oxidative metabolism, or exogenous, both chemicals and physical¹. Among the most harmful lesions, double-strand breaks (DSBs) play a fundamental role in contributing to genomic instability, since they cause chromosome aberrations that in turn can initiate the carcinogenesis process. Thus, cells are provided with complex and efficient mechanisms of DSBs repairing².

When a DSB occurs, the cell triggers the DNA damage response (DDR) where, together with the MRE11/RAD50/NBS1 complex, ATM or ATR k....

Protocol

The study was approved by the ethical committee of Pisa University, and informed and signed consent was obtained from each donor.

1. Formation of γH2AX and 53BP1 foci

1. Preparation of samples and mutagenic treatment
   1. Collect whole blood samples by venipuncture from healthy adult individuals in blood collection (e.g., Vacutainer) tubes containing lithium heparin as an anticoagulant.
   2. In order to guarantee proper blood sample preservation, start the procedure within 24 h of sampling.
   3. Add 300 µL of the sample to a tube containing 4.7 mL of complete medium (0.5% penic....

Results

Data obtained by the fluorescence microscope analysis of peripheral lymphocytes allow us to evaluate three main aspects: the effectiveness of bleomycin treatment in increasing the number of γH2AX and 53BP1 foci (and thus of DSBs) due to its mutagenic effect, at what extent both foci co-localized at the site of DSBs, and the time-course of γH2AX and 53BP1 foci to delineate the repair kinetics of bleomycin-induced DSBs. As expected, a very higher frequency of both γH2AX and 53BP1 foci was observed between un.......

Discussion

Immunofluorescence analysis of γH2AX and 53BP1 foci is a suitable method for assessing genomic damage in interphase nuclei of a cell system. This procedure has several critical points that can affect the outcome of the experiments, mainly, the agents used in fixation and permeabilization, the type of antibodies and their dilution factors, and the concentration of the mutagen.

The maintenance of protein integrity is fundamental since the immunofluorescence method expects to identify antige.......

Materials

Name	Company	Catalog Number	Comments
AlexaFluor 568 goat anti-mouse IgG (γ1)	Invitrogen	A21124	53BP1 secondary antibody
Bleoprim	Sanofi		bleomycin sulfate (mutagen)
Penicillin-streptomycin solution 100X	Euroclone	ECB3001D	antibiotics for culture medium
PBS 10X	Termofisher	14200075	Phosphate-buffered saline
FBS	Euroclone	EC20180L	Fetal Bovine Serum for immunofluorescence
Goat anti-rabbit IgG (H+L) DyLight 488 Coniugated	Termofisher	#35552	γH2AX secondary antibody
Mouse anti-53BP1 monoclonal antibody	Merck	MAB 3802	53BP1 primary antibody
Labophot 2	Nikon		Fluorescence microscope
P-histone H2AX (Ser139) rabbit antibody	Cell Signaling	#2577	γH2AX primary antibody
Phytohemoagglutinin	Termofisher	R30852801	component of culture medium
Prolong gold antifade reagent with DAPI	Cell Signaling	#8961	Antifade solution with DAPI for counterstaining
RPMI 1640	Euroclone	ECB9006L	Culture medium
Triton-X100	Sigma	T9284	Nonionic detergent for permeabilization