Immunofluorescence-Based Assay for Detection of Nuclear RAD51 Foci as a Marker of Homologous Recombination Repair in Ovarian Cancer Cells

Summary

RAD51 nuclear foci are a marker of homologous recombination (HR) activity. This manuscript describes a simple immunofluorescence method to detect these foci in cancer cells, enabling assessment of HR function and supporting preclinical investigation of responses to DNA-damaging agents.

Abstract

RAD51 is a central protein in the homologous recombination (HR) pathway and is essential for the accurate repair of DNA double-strand breaks (DSBs). Following DSB formation, DNA end resection generates single-stranded DNA substrates that facilitate the recruitment and assembly of RAD51 nucleoprotein filaments at sites of damage. This process results in the formation of discrete nuclear RAD51 foci, which serve as a widely accepted functional readout of HR activity and a surrogate marker of HR proficiency. Because defects in HR are common in several malignancies, particularly ovarian and breast cancers, assessment of RAD51 foci formation has emerged as an important approach for evaluating DNA repair capacity and predicting response to DNA-damaging therapies, including platinum compounds and poly(ADP-ribose) polymerase (PARP) inhibitors, whose efficacy is strongly influenced by HR repair status. This manuscript describes a simple, reliable, and reproducible immunofluorescence-based protocol for the detection and quantification of RAD51 nuclear foci in cultured ovarian cancer cells. The method involves induction of DNA damage by ionizing radiation (IR), followed by fixation, immunostaining with antibodies against RAD51 and γH2AX, confocal microscopy, and manual quantitative analysis of RAD51/γH2AX co-localized foci. The protocol can be applied under basal conditions or after genetic and pharmacological perturbations to determine their effects on HR function. Representative results demonstrate robust induction of RAD51 foci in HR-proficient ovarian cancer cells following DNA damage, whereas RAD51 depletion markedly reduces foci formation despite comparable levels of DSBs, confirming assay specificity. Overall, this protocol provides a robust and reproducible functional assay for assessing HR competency, with broad applications in preclinical and potentially translational cancer research.

Introduction

DNA double-strand breaks (DSBs) are among the most severe forms of DNA damage because inaccurate repair can compromise genome stability and contribute to tumor development or cell death^1,2,3. To counteract these threats, cells employ several DNA repair pathways, including homologous recombination (HR), which is considered the most accurate mechanism for repairing DSBs^2,4,5. Unlike error-prone repair pathways, HR utilizes a homologous DNA sequence as a template to restore genetic info....

Protocol

All materials and reagents used in this protocol are listed in the Table of Materials, including manufacturer information, catalog numbers, and antibody dilutions. An overview of the protocol is shown in Figure 1.

NOTE: The ovarian cancer cell lines OVCAR-8 and ABT^R2 are used in this protocol and have been previously described^{15,16,17,18,19,20}. OVCAR-8 cells were kindly provided by Dr. La....

Results

IR induces RAD51 foci formation at DNA DSBs in OVCAR-8 cells
To functionally evaluate HR activity, RAD51 foci formation was examined in HR proficient OVCAR-8 ovarian cancer cells following induction of DNA DSBs by IR. To establish assay specificity and confirm that observed foci represent bona fide RAD51-dependent HR events, cells were transfected with either non-targeting control luciferase siRNA (siLUC) or RAD51-targeting siRNA (siRAD51.)¹⁵

Discussion

The immunofluorescence-based detection of nuclear RAD51 foci described here provides a reliable and functional approach to assess HR activity in cultured ovarian cancer cells. Unlike genomic or transcriptomic analyses that infer HR status indirectly, RAD51 foci formation directly reflects the cell’s ability to recruit and assemble the core HR machinery at sites of DNA DSBs. As such, this assay serves as a sensitive readout of HR competency following DNA damage and is well-suited for mechanistic studies investigatin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Polyacylamide gel recipe (Lower gel buffer 20 ml,ddH2O 33.4 mL, 30% Acrylamide/Bis-acrylamide (29:1) 26.6 mL. 10% APS (fresh) 200 µL  TEMED 70 µL)	Home-made	NA	Resolving gel for SDS-PAGE
10% Polyacylamide gel recipe (Upper gel buffer 10 mL, ddH2O 23.6 mL, 30% Acrylamide/Bis-acrylamide (29:1) 6.4 mL 10% APS (fresh) 160 µL  TEMED 40 µL)	Home-made	NA	Stacking geL for SDS-PAGE
100 mL Trypsin EDTA 1X, 0.25% Trypsin/2.21 mM EDTA in Sodium Bicarbonate	Corning	25-053-C1	Used to detach adherent cells during routine passaging and prior to cell seeding.
10x Phosphate Buffered Saline (PBS)	Bio-Rad	1610780	Diluted to 1× and used for washing steps, blocking solution, antibody dilutions, fixation, and permeabilization.
10X Running buffer (6 L) Tris 181.8 g +Glycene 864.0 g+SDS 60 g	Home-made	NA	Run to proteins sample in the gel
10X Transfer (6 L) Tris 181.8 g+ Glycene 864 g	Home-made	NA	Transfter the protein from gel to membrane
10x Tris Buffered saline	Bio-Rad	1706435	buffer
30%  (w/v)Acrylamide ;0.8(w/v)/Bis-acrylamide stock solution (37.5:1)	Ultrapure EC-890	EC-890	For SDS-PAGE
ABTR2 cells	Mayo clinic	NA	acquired PARPi-resistant ovarian cancer cell lines
AKT Lysis Buffer Recipe (50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM 2-glycerophosphate, 10 μg/mL leupeptin, 5 μg/mL aprotinin, 5 μg/mL pepstatin, and 20 mM microcystin-LR.	Home-made	NA	Cell lysis buffer
Ammonium Persulfate	Bio-Rad	16107000	Molecular biology as a polymerization for gels an oxidizing agent used as a free-radical initiator
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301	Sigma-Aldrich	05-636	Primary antibody used to detect γH2AX foci. Used at 1:1000 dilution
Anti-Rad51 (Ab-1) Rabbit pAb	Sigma-Aldrich	PC130	Primary antibody used to detect RAD51 nuclear foci as a marker of homologous recombination activity. Used at 1:1000 dilution
Bovine serum albumin Protease free	GoldBio	A-420-250	Used to prepare blocking solution (5% BSA in PBS) to reduce non-specific antibody binding during immunofluorescence staining.
ChemiDoc Imaging System	Bio-Rad	12003154	Imaging system
Confocal Laser Scanning Microscope	ZEISS	Zeiss LSM 780	Immunofluorescence imaging, γH2AX foci analysis, RAD51 foci quantification, protein co-localization, and 3D image acquisition
Countess II FL Automated Cell Counter	Life Technologies	AMQAF1000	Automated Cell Counter
Cover slips	Cardinal Health	M6045-10A	Placed over mounted samples to protect stained cells and enable high-resolution imaging.
ECM 830 Square Wave Electroporation System	Hbio-BTX	45-2052	Electroporation system suitable for applications for gene delivery in mammalian cells
Fetal Bovine Serum	Corning	35-010-CV	Added to RPMI-1640 (as 8%) to support cell growth and viability.
Glycine	Thermo Fisher Scientific	A13816.0C	Buffer
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594	Thermo Fisher Scientific	A-11032	Secondary antibody used to detect γH2AX primary antibody. Used at 1:350 dilution
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Thermo Fisher Scientific	A-11034	Secondary antibody used to detect RAD51 primary antibody. Used at 1:350 dilution
GraphPad Prism	GraphPad Software	version 10.6.1	Statistical analysis, graph generation, and data visualization.
HEPES 1M	Corning	25060-C1	PH maiantance buffer in the pH 7.2-7.6 range
Hoechst 33342 solution (20 mM, 5 mL)	Thermo Fisher Scientific	62249	was used for nuclear staining
Horseradish peroxidase-conjugated anti-mouse immunoglobulin G	Cell Signaling Technology	7076S	Secondary antibody used for detection of mouse (β-actin) primary antibody in Western blotting.
Horseradish peroxidase-conjugated anti-rabbitimmunoglobulin G	Cell Signaling Technology	7074S	Secondary antibody used for detection of rabbit primary antibody (RAD51) in Western blotting.
Instant non-fat dry milk	Hyvee	G461A323	Blocking reagent
Laemmli Sample Buffer (4×)	Bio-Rad	1610747	Sample Buffer
Lonza's MycoAlert Mycoplasma Detection Assays	Lonza Bioscience	LT07-218	Used to routinely confirm that cell cultures are free of mycoplasma contamination.
Lower gel Recipe is (1 L)(Tris 181.7 g+SDS 4.0 g)PH to 8.8 w/cenc HCl	Home-made	NA	Resolving gel for SDS-PAGE
Mouse monoclonal Anti-beta Actin antibody	Abcam	ab8226	Primary antibody used to detect β-actin as a loading control in Western blot analyses.
Non-targeting control luciferase siRNA (siLUC): 5′-CUUACGCUGAGUACUUCGA-3′	Horizon Discovery	NA	working concentration 20 nmol
Nunc Lab Tek II, 8-chamber slide W/cover RS Glass slide sterile	Thermo Fisher Scientific	154534	Used for seeding and culturing cells for immunofluorescence staining and confocal imaging.
OVCAR-8 cells	National Cancer Institute, (NIH)	NA	Ovarian cancer cell lines
Paraformaldehyde 16% Aqueous Solution EM Grade	Electron microscopy sciences	15710-S	Diluted to 3% and used to fix cells prior to immunofluorescence staining.
Prolong Gold antifade Mountant	Thermo Fisher Scientific	P36930	Used to mount samples and preserve fluorescence signals during confocal imaging.
Protease inhibitor	Roche / Sigma-Aldrich	11836170001	Protease Inhibitor Cocktail
Protein Assay dye reagent concentration	Bio-Rad	5000006	Protein quantification
PVDF membrane (Immobilon-P .45 µm)	Millipore	IPVH00010	Transfer membrane
RAD51 siRNA (siRAD51): 5′-GGGAUUUGUGAAGCCAAA-3	Horizon Discovery	NA	working concentration 20 nmol
RPMI-1640 (with L-glutamine and glucose)	Corning	10-041-CV	Used as the basal growth medium for culturing ovarian cancer cell lines (OVCAR-8 and ABTR2).
Sodium Chloride (NaCl)	Fisher scientific	S271-500	Buffer
Sodium dodecyl sulfate (SDS)	Bio-Rad	1610302	Buffer
Sodium Fluoride	Sigma-Aldrich	S7920	Phosphatase inhibitor
Sodium orthovanadate	Sigma-Aldrich	S6508-50G	Phosphatase inhibitor
Sodium pyrophosphate decahydrate	Sigma-Aldrich	221368	Buffer
Sodium β-Glycerophosphate Pentahydrate	Thermo Scientific Chemicals (formerly Alfa Aesar)	L03425.22	Phosphatase inhibitor
SuperSignal™ West Pico PLUS Chemiluminescent Substrate	Thermo Fisher Scientific	34580	Detection system
TEMED 50 mL	Bio-Rad	1610801	Molecular biology as a polymerization catalyst for polyacrylamide gels with help of APS 10%
Topcoat rapid dry clear nail polish	Electron microscopy sciences	72180	Used to seal coverslip edges and prevent drying or movement of mounted samples.
Triton x-100	Bio-Rad	1610407	Used for permeabilization of fixed cells to allow antibody access to nuclear proteins.
Tween-20	Sigma-Aldrich	P1379-1L	Buffer
UltraPure EDTA	Thermo Fisher Scientific	15576028	Chelator of divalent metal cations. It is suitable for biochemistry or molecular biology applications
Upper gel Recipe is (1 L) (Tris 60.6 g+ SDS 4.0 g)PH to 6.8 w/ conc HCl	Home-made	NA	Stacking geL for SDS-PAGE
Zeiss LSM 780 laser scanning confocal microscope	Carl Zeiss, Germany	NA	Used to acquire high-resolution confocal images for visualization and quantification of RAD51 and γH2AX nuclear foci.
β-Mercaptoethanol	Bio-Rad	1610710	Reducing agent