Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Summary

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of γH2AX and 53BP1.

Abstract

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of γH2AX and 53BP1, the combined analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on γH2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the γH2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of γH2AX and 53BP1.

Introduction

DNA is continuously damaged by endogenous (e.g., replication stress, reactive oxygen species, intrinsic instability of DNA) and exogenous (e.g., chemical radicals, irradiation) sources (Figure 1)^1,2,3,4. Among DNA damage, DNA double-strand breaks (DSB) are particularly serious lesions and may induce cell death or carcinogenesis. About 50 DSB may arise per cell and cell cycle⁵. In mammalian cells, homologous recombination (HR) and nonhomologous end-joining (NHEJ) developed as major pathways for the repair of DSB (Figure 2). HR occurs in late S/G2 phase and uses an intact sister chromatid as a template for potentially error-free repair. In comparison, NHEJ is active throughout the cell cycle and potentially mutagenic as base pairs may be added or resected before ligation of the broken ends. In addition, alternative end-joining may be engaged as a slow mutagenic back-up repair mechanism in case of NHEJ deficiency^6,7.

DSB induce the phosphorylation of the histone H2AX at Serine 139 in a region of several megabase pairs around each DSB. The forming nuclear foci are named γH2AX foci and are detectable by immunofluorescence microscopy⁸. γH2AX promotes the recruitment of further DSB-responsive proteins and is involved in chromatin remodeling, DNA repair, and signal transduction⁹. As each γH2AX focus is considered to represent a single DSB, the quantification of DSB by immunofluorescence microscopy is possible, which has been demonstrated in cancer cell lines and patient specimens^{10,11,12,13,14}. 53BP1 (p53 binding protein 1) is another key protein in mediating DSB repair. It is involved in the recruitment of DSB-responsive proteins, checkpoint signaling, and the synapsis of DSB ends¹⁵. In addition, 53BP1 plays a critical role in the DSB repair pathway choice. It triggers the repair of DSB towards NHEJ while HR is prevented¹⁶. Considering the genuine functions of γH2AX and 53BP1 in DSB repair, the simultaneous analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a useful method for the precise analysis of the formation and repair of DSB.

This manuscript provides a step-by-step protocol for performing immunofluorescence microscopy of γH2AX and 53BP1 in cell nuclei. Specifically, the technique is applied in normal fibroblasts of the cell line NHDF, in x-ray irradiated lymphocytes of a healthy individual and in CD34+ cells of a patient with acute myeloid leukemia. The details of the method are pointed out in the context of the presented results.

Protocol

All methods described here have been approved by the Ethics Committee II of the Medical Faculty Mannheim of the Heidelberg University. Written informed consent was obtained from all individuals.

1. Preparation of Materials

1. Anticoagulant stock solution: Prepare an anticoagulant stock solution of 200 I.U. heparin per mL in 0.9% sodium chloride. Fill each of the collection tubes (draw volume 9 mL) with 2 mL of the anticoagulant stock solution before withdrawal of the blood or bone marrow samples.
2. Lysis solution for red cells: Prepare the 10x lysis solution for red cells with 82.91 g ammonium chloride, 7.91 g ammonium bicarbonate, and 2 mL of 0.5 M ethylenediaminetetraacetic acid solution to a pH of 8.0 by dissolving the agents in double-distilled water for a total volume of 1 L.
3. Fixation solution: Add 8.3 µL of a 1 M potassium hydroxide solution to 360 mg paraformaldehyde (PFA) in a microtiter tube. Fill the tube with phosphate buffered saline (PBS) to the 1 mL mark of the tube and heat the tube in a heating block at 95 °C to get 1 mL of a 36% PFA solution. Transfer the 1 mL 36% PFA solution to a tube with a capacity of 15 mL. Add 8 mL PBS to the 1 mL 36% PFA solution, to get 9 mL of a 4% PFA stock solution. Aliquot the 4% PFA stock solution and store at -18 °C until use for fixation of the cells.
   CAUTION: 1) Potassium hydroxide solution is corrosive. Be aware of eye and skin protection. 2) PFA is carcinogenic. Avoid skin contact and inhalation. Prepare the fixation solution under a hood.
4. Permeabilization solution: Add 50 µL Octoxinol 9 to 50 mL PBS to get a solution of approximately 0.1% Octoxinol 9.
5. Blocking solution: Prepare 5% and 2% blocking solutions by mixing the protein blocking agent with PBS and store at 6 – 8 °C.

2. Preparation of the Samples

1. Fibroblasts in cell culture: Cultivate human fibroblasts of the cell line NHDF in a humidified 5% CO₂ atmosphere at 37 °C in RPMI 1640 medium supplemented with 10% fetal calf serum, 4 mM glutamine, and 1% penicillin/streptomycin.
   1. Preparation of a microscope slide with adherent fibroblasts
      1. Remove the medium from the flask (75 cm² growth area) with exponentially growing NHDF fibroblasts. Add 10 mL PBS to the flask. Wash the adherent fibroblasts gently by manually shaking the flask for 1 min.
      2. Remove the PBS and add 1 mL trypsin into the flask. Shake the flask gently to ensure that all adherent cells are covered by trypsin. Remove the trypsin from the flask. Put the flask into the incubator for 5 min at 37 °C.
      3. Take the flask out of the incubator and add 10 mL RPMI 1640 medium into the flask. Pipette the medium up and down several times to disperse the fibroblasts.
      4. Pipette 0.3 mL of the dispersed fibroblasts onto each of the two fields of the microscope slide and put the slide into the incubator for 24 h so that the fibroblasts adhere to the slide. For immunostaining of γH2AX and 53BP1 in nuclei of fibroblasts, move on to step 3.1.
2. Patient samples
   1. Blood samples: Use the collection tubes that were filled with 2 mL of the anticoagulant stock solution and add 7 mL blood into the tubes. Store the samples overnight at room temperature (i.e., 18 - 20 °C). The next day, isolate the G0/G1 phase resting mononuclear cells by density gradient centrifugation (step 2.2.3).
   2. Bone marrow samples: Use the collection tubes that were filled with 2 mL of the anticoagulant stock solution (step 1.1) and add 7 mL bone marrow into the tubes. Store the samples overnight at room temperature (i.e.,18 - 20 °C). The next day, isolate the G0/G1 phase resting mononuclear cells by density gradient centrifugation (step 2.2.3). Use CD34 microbeads and cell separation columns for the isolation of the CD34+ cells.
   3. Isolation of mononuclear cells by density gradient centrifugation
      NOTE: Mononuclear cells are isolated from blood and bone marrow samples by density gradient centrifugation^17,18,19.
      1. Dilute the heparinized blood sample in a ratio of 1:1 with PBS and the heparinized bone marrow sample in a ratio of 1:3 with PBS. Suspend the cells gently by drawing them in and out of the pipette two times.
      2. Add a volume of density gradient medium to a fresh tube. Gently layer the diluted blood or bone marrow on top of the density gradient medium. Take care not to mix the two layers.
      3. Centrifuge the tube for 30 min at room temperature and 400 x g. Draw the upper plasma layer off with the pipette. Then carefully harvest the mononuclear cells in the layer above the density gradient medium. Transfer the mononuclear cells into a fresh tube.
      4. Add at least three volumes of PBS to the mononuclear cells in the tube. Suspend the cells gently by drawing them in and out of the pipette two times. Centrifuge the tube for 10 min at room temperature and 400 x g.
      5. After the spin remove the supernatant and add 10 mL of 4 °C, 1x lysis solution for red cells. Resuspend the cells gently by drawing them in and out of the pipette two times, and place the tube on ice for 5 min. Then add 30 mL PBS at 4 °C and centrifuge the tube for 10 min at room temperature and 400 x g.
   4. Preparation of the cytospins
      1. Prepare two cytospins with mononuclear cells of the patient samples (i.e., one cytospin for γH2AX immunofluorescence staining and one cytospin for γH2AX and 53BP1 combined immunofluorescence staining) by centrifugation (300 x g, 10 min) of 1.0 x 10⁵ cells for each preparation (Figure 3 and 4).

3. γH2AX and 53BP1 Immunofluorescence Staining

1. Fixation and permeabilization: Fix the cells with 200 µL of 4% PFA for 10 min. After fixation, wash the cells gently three times with 30 mL of PBS for 5 min each on a lab shaker. Then permeabilize the cells with 200 µL of 0.1% Octoxinol 9 for 10 min. Wash the cells gently three times with 30 mL of 5% blocking solution for 5 min each on a lab shaker. Block the cells in 30 mL of fresh 5% blocking solution for 1 h.
2. Incubation with antibodies
   1. Incubate one preparation of the cells with a mouse monoclonal anti-γH2AX antibody (1:500) and the other preparation of the cells with a mouse monoclonal anti-γH2AX antibody (1:500) and a polyclonal rabbit anti-53BP1 antibody (1:500) overnight at 4 °C.
   2. After incubation wash the cells gently 3 times with 30 mL of 2% blocking solution for each 5 min on a lab shaker.
   3. Remove the blocking solution and incubate the first preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and the second preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and an Alexa555-conjugated donkey anti-rabbit secondary antibody (1:500) for 1 h at room temperature.
3. Mounting medium: Put the slide with the cells in a bowl and wash the cells gently three times with 30 mL of PBS for each 5 min on a lab shaker. Remove the PBS and mount the cells with mounting medium containing 4,6-diamidino-2-phenylindole. Cautiously put a coverslip on top of the mounting medium so that no air bubbles are embedded. Wait at least 3 h for hardening of the mounting medium before analyzing the cells by fluorescence microscopy.

4. Analysis of γH2AX and 53BP1 Foci

1. Fluorescence microscopy: Analyse the γH2AX and 53BP1 foci in the cell nuclei with a fluorescence microscope equipped with filters for DAPI, Alexa 488, and Cy3 during imaging at a 100X objective magnification. Record images with a camera and process the images with appropriate imaging software.

Results

Analysis of γH2AX foci in cells is most accurate in the G0/G1 phase and the G2 phase when γH2AX foci appear as distinct fluorescent dots (Figure 5A). In contrast, analysis of γH2AX foci in cells during the S phase is complicated by dispersed pan-nuclear γH2AX speckles caused by the replication process (Figure 5B).

Fixation of the cells was performed ...

Discussion

Immunofluorescence microscopy of γH2AX and 53BP1 is a useful method for analyzing formation and repair of DSB in a broad spectrum of research areas. Critical parameters that influence the outcome of the experiments are the phase of the cell cycle, the agents used for the fixation and permeabilization of the cells, the choice of the antibodies, and the hardware and software of the fluorescence microscope.

The influence of the cell cycle on γH2AX foci patterns was demonstrated by expon...

Materials

Name	Company	Catalog Number	Comments
RPMI medium	Sigma-Aldrich	R0883	Medium for cell culture
Heparin sodium	ratiopharm	PZN 3029843	Heparin 5,000 I.U. / mL
Sodium chloride solution 0.9%	B. Braun	PZN 1957154	Component of the anticoagulant stock solution
Ficoll-Paque Premium	GE Healthcare	17-5442-02	Medium for isolation of mononuclear cells
Trypsin solution 10X	Sigma-Aldrich	59427C	Enzyme for dissociation of fibroblasts in cell culture
CD34 MicroBead Kit	Miltenyi Biotec	130-046-702	Isolation of CD34+ myeloid progenitor cells
Diagnostic microscope slides	Thermo Scientific	ER-203B-CE24	Microscope slides
Megafuge 1.0 R	Heraeus	75003060	Tabletop centrifuge
Cytospin device
Lid	Heraeus	76003422	Lid for working without micro-tubes
Cyto container	Heraeus	75003416	Cyto container with 2 conical bores
Clip carrier	Heraeus	75003414	Carrier for holding a cyto container and a slide
Support insert	Heraeus	75003417	Support insert for holding a clip carrier
Triton X-100 (Octoxinol 9)	Thermo Scientific	85112	Detergent for permeabilization of cell membranes
Potassium hydroxide solution 1M	Merck Millipore	109107	Necessary for preparing the paraformaldehyde solution
Paraformaldehyde	Sigma-Aldrich	P6148	Fixation agent
Phosphate buffered saline	Sigma-Aldrich	D8537	Balanced salt solution
Chemiblocker	Merck Millipore	2170	Blocking agent
Mouse monoclonal anti-γH2AX antibody (JBW301)	Merck Millipore	05-636	Primary antibody for detection of γH2AX
Polyclonal rabbit anti-53BP1 antibody (NB100-304)	Novus Biologicals	NB100-304	Primary antibody for detection of 53BP1
Alexa Fluor 488-conjugated goat anti-mouse antibody	Invitrogen	A-11001	Secondary antibody
Alexa Fluor 555-conjugated donkey anti-rabbit antibody	Invitrogen	A-31572	Secondary antibody
Vectashield mounting medium	Vector Laboratories	H-1200	Contains DAPI for staining of DNA
Axio Scope.A1	Zeiss	490035	Fluorescence microscope
Cool Cube 1 CCD camera	Metasystems	H-0310-010-MS	Camera system for digital recording
Isis software	Metasystems	Not applicable	Microscope software