Axioobserver z1 inverted microscope

Cells were pulse labeled with 25 μM CldU followed by 250 μM IdU for 20–45 min each. Labeled cells were trypsinized, lysed in spreading buffer (200 mM Tris-Hcl pH 7.4, 50 mM EDTA, and 0.5% SDS) and spread on microscope slide (Menzel-Gläser, Superfrost). DNA fibers were fixed on slides using 3:1 methanol: acidic acid. Slides were treated with 2.5 M HCl for 1 h and 15 min to denature DNA followed by 1 h incubation in blocking buffer (PBS, 1% BSA, 0.1% Tween20) to block background staining. For detection of CldU and IdU, slides were incubated for 1 h with rat-anti-Brdu (Clone BU1/75, Abcam; 1:500) and mouse-anti-BrdU (clone B44, Becton Diskinson; 1:750), respectively. Subsequently, slides were fixed with 4% paraformaldehyde for 10 min and incubated with Alexa 488-labeled goat-anti-mouse and Alexa 555-labeled goat-anti-rat (Molecular probes; 1:500) for 1 h and 30 min. DNA fibers were imaged with the Zeiss AxioObserver Z1 inverted microscope using a 63x objective equipped with a Hamamatsu ORCA AG Black and White CCD camera. Replication tracks lengths were analyzed using ImageJ software and the conversion factor 1 μm = 2.59 kb was used.

Cells were pulse-labelled with 25 μM CldU followed by 250 μM IdU for 20–40 min each. After labelling, cells were trypsinized and lysed in spreading buffer (200 mM Tris-Hcl pH 7.4, 50 mM EDTA and 0.5% SDS) before spreading on a microscope slide (Menzel-Gläser,Superfrost). Slides were fixed in methanol: acidic acid 3:1. Before immunodetection, slides were treated with 2.5 M HCl for 1 hr and 15 min. To detect CldU and IdU labelled tracts slide were incubated for 1 hr with rat-anti-Brdu (Clone BU1/75, Novus Biologicals; 1:500) and mouse-anti-BrdU (clone B44, Becton Diskinson; 1:750), respectively. Subsequently, slides were fixed with 4% paraformaldehyde for 10 min and incubated with Alexa 488-labeled goat-anti-mouse and Alexa 555-labeled goat-ant-rat (Molecular probes; 1:500) for 1 hr and 30 min. Pictures were taken with a Zeiss AxioObserver Z1 inverted microscope using a 63x lens equipped with a cooled Hamamatsu ORCA AG Black and White CCD camera and track lengths were analyzed with ImageJ software. Replication track lengths were calculated using the conversion factor 1 μm = 2.59 kb (Jackson and Pombo, 1998 (link)). The 1-way ANOVA (nonparametric Kruskal-Wallis test) was used for statistical analyses.

Neutral comet assays were performed as described by Olive and Banath (24 (link)). Briefly, 8 × 10³ cells were diluted in 0.4 ml of PBS and were added to 1.2 ml of 1% low-gelling-temperature agarose (Sigma). Subsequently, the cell suspension was transferred onto precoated slides (Menzel-Gläser). Cell lysis was performed in neutral lysis solution (2% sarkosyl, 0.5 M Na₂EDTA, 0.5 mg/ml proteinase K) at pH 8.0 overnight at 37°C. Slides were washed 3 times with neutral rinse and electrophoresis buffer (90 mM Tris, 90 mM boric acid, 2 mM Na2EDTA) at pH 8.5, and electrophoresis was performed in neutral rinse and electrophoresis buffer for 25 min at 20 V. Nuclei were stained with 2.5 mg/ml propidium iodide (Invitrogen) in distilled water for 20 min. Pictures of individual cells were taken with a Zeiss AxioObserver Z1 inverted microscope equipped with a cooled Hamamatsu ORCA AG black-and-white CCD camera, and were analyzed with CASP software (http://www.casplab.com). P-values were determined using an unpaired t-test with Welch's correction.