Materials and Reagents

60 mm cell culture dishes (Sigma-Aldrich, catalog number: CLS430166 )
Manufacturer: Corning, catalog number: 430166.
Silane-Prep Slides: glass slides coated with silane (aminoalkylsilane) (Sigma-Aldrich, catalog number: S4651 )
HeLa cells (ATCC, catalog number: CCL-2 )
Cell culture
1. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 61870010 )
2. 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
3. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070063 )
4. 0.25% trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056 )
Note: HeLa cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin (defined as ‘complete medium’).
5-Iodo-2’-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125 )
5-Chloro-2’-deoxyuridine (CldU) (Sigma-Aldrich, catalog number: C6891 )
Chemicals of analytical grade

Methanol (CARLO ERBA Reagents, catalog number: 414814 )
Acetic acid glacial (Fisher Scientific, catalog number: A38-212 )
Ethanol absolute (CARLO ERBA Reagents, catalog number: 308605 )
39.5% hydrochloric acid (HCl) (CARLO ERBA Reagents, catalog number: 403878 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
Tween^® 20 (Sigma-Aldrich, catalog number: P9416 )
ProLong^® Gold anti-fade reagent (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36935 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 255793 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: 795488 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71725 )
Tris (Roche Diagnostics, catalog number: 10708976001 )
0.5 M EDTA (pH 8.0) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9262 )
NP-40 (Thermo Fisher Scientific, catalog number: 85124 )

Antibodies

Anti-BrdU clone BU1/75 (ICR1) (detects CldU) (Bio-Rad Laboratories, catalog number: OBT0030CX )
Anti-BrdU clone B44 (detects IdU) (BD, BD Biosciences, catalog number: 347580 )
Alexa Fluor 488-conjugated chicken anti-rat (Thermo Fisher Scientific, Invitrogen, catalog number: A21470 )
Alexa Fluor 594-conjugated rabbit anti-mouse (Thermo Fisher Scientific, Invitrogen, catalog number: A-11062 )

1x phosphate buffered saline (PBS) (see Recipes)
Spreading buffer (see Recipes)
Stringent buffer (see Recipes)

Equipment

Incubator
UVC 500 UV Crosslinker (GE Healthcare, model: UVC 500 )
Centrifuge
Confocal microscope (e.g., Carl Zeiss, model: Zeiss LSM100 )
Fume hood

Software

GraphPad software

Procedure

See Figure 1 for an overview of the protocol.

Figure 1. Schematic overview of the protocol

Culture HeLa cells in RPMI 1640 medium supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin (defined as ‘complete medium’).
Treat a 60 mm dish of 80% confluent HeLa cells with CldU (20 μM final concentration) in complete RPMI 1640 medium (these cells are defined as ‘labelled cells’); whereas leave untreated another 60 mm dish of the same cells in the same conditions (these cells are defined as ‘unlabelled cells’). Incubate both dishes at 37 °C in a 5% CO₂ incubator for 20 min (see Note 1).
Wash twice with 1x PBS.
Remove all the excess 1x PBS.
Treat labelled HeLa cells with UVC at a dose of 10 J/m² (see Note 2).
Add to labelled cells IdU (100 μM final concentration) in fresh complete RPMI 1640 medium (see Note 1).
Incubate at 37 °C in a 5% CO₂ incubator for 40 min.
Trypsinize both labelled and unlabelled HeLa cells, then harvest them through centrifugation (800 x g for 5 min).
Wash the cell pellet with ice-cold (4 °C) 1x PBS twice.
Resuspend HeLa cells with the ice-cold 1x PBS at 2.5 x 10⁵ cells/ml.
Dilute labelled cells 1:8 with unlabelled cells (vol/vol).
Spot onto the Silane Prep slides 2.5 μl of this labelled/unlabelled cell solution. Allow the 2.5 μl sample to evaporate not completely. (see Note 3)
Mix the cells with 7.5 μl of spreading buffer directly on the Silane Prep slide.
Incubate at room temperature (RT) for 10 min.
Tilt the slides to 15° to spread DNA fibers along their length.
Air-dry the DNA spreads. (see Note 3)
Fix DNA fibers (which are now spread onto the Silane Prep slide) in 3:1 methanol/acetic acid in a plastic or glass container at -20 °C for 15 min.
Wash slides in 1x PBS twice.
Incubate DNA fibers in 70% ethanol at 4 °C for one hour (see Note 4).
Treat the slides with 2.5 N of HCl at 37 °C for one hour.
Wash slides with 1x PBS twice.
Block DNA fibers with 1x PBS + 0.5% BSA for 30 min at RT.
Incubate slides with 1:500 Anti-BrdU clone BU1/75 (ICR1) (which detects CldU) and 1:300 Anti-BrdU clone B44 (which detects IdU) together in 1x PBS + 0.5% BSA for one hour at 37 °C.
Wash DNA fibers twice for 5 min at RT and once for 15 min at RT with stringent buffer (no shaking required).
Incubate Silane Prep slides with 1:300 Alexa Fluor 488-conjugated chicken anti-rat and 1:400 Alexa Fluor 594-conjugated rabbit anti-mouse together in 1x PBS + 0.5% BSA for 30 min at 37 °C. Both antibodies were tested singularly to detect possible cross reactivity in HeLa cells and were previously successfully used for this technique (Alfano et al., 2016).
Wash Silane Prep slides with 1x PBS + 0.5% Tween 20 for 5 min at RT for three times.
Mount the slides with ProLong Gold anti-fade reagent.
Analyse the slides through confocal fluorescent microscopy in sequential scanning mode.

Data analysis

To process the images obtained from the DNA fiber assay (Figure 2) one can use either manual counting or an automatic analysis by using a specific software. To analyze the frequency of the various DNA fiber types (ongoing, newly fired, or others) we manually counted 600 fibers for each experiment of three independent ones. To evaluate the statistical significance among different conditions or treatments assessed through DNA fiber assay, we applied the appropriate statistical test through the GraphPad software. To measure the length of DNA spreads to estimate the replication speed (1 μm corresponds to 2.59 kb), we used the specific image analysis software ImageJ (https://imagej.nih.gov/ij/). The University of North Carolina developed a software for the automatic analysis of DNA spreads (http://dnafiberanalysis.com/).

Figure 2. Representative kinds of DNA fibers identified by using this protocol. The sequential labelling provides a snapshot of different DNA replication steps: 1) Ongoing forks can appear as in a (in the case of unidirectional progression through the replication fork) or as in b (in the case of bidirectional progression through the replication fork). 2) Termination events can appear as in a (as an only-green line in the case of a replication origin in which DNA synthesis terminated while the cells were still undergoing incubation with the first label), or as in b (as adjoined red-green-red signals with divergent trend). 3) New fired origins appear as shown in the lower panel of the figure. These origins were fired during incubation with the second label (consisting of an only-red line, according to the protocol herein described).

Notes

The pulse time and the working concentration of CldU and IdU might have to be adapted depending on the different type of cells under study.
Prior to exposing the cells to the UV bulb remove the plate cap and make sure to aspirate completely any residual cell growth medium or 1x PBS so as not to shield the UV radiations.
Do not evaporate the samples in the fume hood.
At this step, the slides can be stored at 4 °C in 70% ethanol overnight, but we prefer to process the fibers directly.

Recipes

1x phosphate buffered saline (PBS)
137 mM NaCl
2.7 mM KCl
10 mM Na₂HPO₄
1.8 mM KH₂PO₄
Adjust to a final pH of 7.4
Spreading buffer
0.5% sodium dodecyl sulfate (SDS)
200 mM Tris-HCl pH 7.4
50 mM EDTA pH 8
Stringent buffer
10 mM Tris-HCl pH 7.4
400 mM NaCl
0.2% Tween 20
0.2% NP-40

Acknowledgments

We are grateful to the Sbarro Health Research Organization (http://www.shro.org), the Human Health Foundation (http://www.hhfonlus.org), the Commonwealth of Pennsylvania and the Associazione Italiana per la Ricerca sul Cancro (IG 2014-15690) for their support. A.G. is also Director of the Cell Cycle and Cancer Research Line at CROM, Istituto Nazionale Per Lo Studio E La Cura Dei Tumori; Naples. F.P. is also Adjunct Associate Professor at Temple University, Department of Biology, Philadelphia, PA, USA.

References

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