Limits...
A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Show MeSH

Related in: MedlinePlus

UV-induced UDS assay in normal human primary fibroblasts. (A) Normal 48BR cells were cultured on coverslips, UVC-irradiated at the indicated doses, followed by incubation with 10 μM EdU for different time periods, fixation and conjugation of fluorescent dye to the incorporated EdU as described in ‘Materials and methods’ section. The intensity of nuclear fluorescence, which is associated with UDS activity, was analysed using a fluorescence microscope and image-processing software. For each data point, at least 250 nuclei were analysed. Points and error bars indicate means of discrete nuclei fluorescent intensity and standard errors, respectively. (B) Typical photos of the EdU assay are shown. 48BR cells were UVC irradiated (20 J/m2, +UV) or mock-treated (−UV), followed by 2-h incubation with EdU. EdU, coupled to Alexa fluor 488-azide; PH, phase contrast. (C–F) UDS performed by incorporation of EdU (C and D) or BrdU (E and F) was compared. XP15BR cells are primary fibroblasts from an XP-A patient. Cells were UVC irradiated (20 J/m2), followed by incubation with 10 μM EdU or 5 μM BrdU for 2 h. Bars represent frequencies of the fluorescence levels in the indicated classes, with (black) or without (white) UVC irradiation. Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and unirradiated samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651789&req=5

Figure 1: UV-induced UDS assay in normal human primary fibroblasts. (A) Normal 48BR cells were cultured on coverslips, UVC-irradiated at the indicated doses, followed by incubation with 10 μM EdU for different time periods, fixation and conjugation of fluorescent dye to the incorporated EdU as described in ‘Materials and methods’ section. The intensity of nuclear fluorescence, which is associated with UDS activity, was analysed using a fluorescence microscope and image-processing software. For each data point, at least 250 nuclei were analysed. Points and error bars indicate means of discrete nuclei fluorescent intensity and standard errors, respectively. (B) Typical photos of the EdU assay are shown. 48BR cells were UVC irradiated (20 J/m2, +UV) or mock-treated (−UV), followed by 2-h incubation with EdU. EdU, coupled to Alexa fluor 488-azide; PH, phase contrast. (C–F) UDS performed by incorporation of EdU (C and D) or BrdU (E and F) was compared. XP15BR cells are primary fibroblasts from an XP-A patient. Cells were UVC irradiated (20 J/m2), followed by incubation with 10 μM EdU or 5 μM BrdU for 2 h. Bars represent frequencies of the fluorescence levels in the indicated classes, with (black) or without (white) UVC irradiation. Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and unirradiated samples.

Mentions: We found that 20 J/m2 UV irradiation followed by 2-h EdU incubation was the optimal condition for the UDS assay. This condition was used for all experiments except for Figures 1A and 5.Figure 1.


A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

UV-induced UDS assay in normal human primary fibroblasts. (A) Normal 48BR cells were cultured on coverslips, UVC-irradiated at the indicated doses, followed by incubation with 10 μM EdU for different time periods, fixation and conjugation of fluorescent dye to the incorporated EdU as described in ‘Materials and methods’ section. The intensity of nuclear fluorescence, which is associated with UDS activity, was analysed using a fluorescence microscope and image-processing software. For each data point, at least 250 nuclei were analysed. Points and error bars indicate means of discrete nuclei fluorescent intensity and standard errors, respectively. (B) Typical photos of the EdU assay are shown. 48BR cells were UVC irradiated (20 J/m2, +UV) or mock-treated (−UV), followed by 2-h incubation with EdU. EdU, coupled to Alexa fluor 488-azide; PH, phase contrast. (C–F) UDS performed by incorporation of EdU (C and D) or BrdU (E and F) was compared. XP15BR cells are primary fibroblasts from an XP-A patient. Cells were UVC irradiated (20 J/m2), followed by incubation with 10 μM EdU or 5 μM BrdU for 2 h. Bars represent frequencies of the fluorescence levels in the indicated classes, with (black) or without (white) UVC irradiation. Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and unirradiated samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651789&req=5

Figure 1: UV-induced UDS assay in normal human primary fibroblasts. (A) Normal 48BR cells were cultured on coverslips, UVC-irradiated at the indicated doses, followed by incubation with 10 μM EdU for different time periods, fixation and conjugation of fluorescent dye to the incorporated EdU as described in ‘Materials and methods’ section. The intensity of nuclear fluorescence, which is associated with UDS activity, was analysed using a fluorescence microscope and image-processing software. For each data point, at least 250 nuclei were analysed. Points and error bars indicate means of discrete nuclei fluorescent intensity and standard errors, respectively. (B) Typical photos of the EdU assay are shown. 48BR cells were UVC irradiated (20 J/m2, +UV) or mock-treated (−UV), followed by 2-h incubation with EdU. EdU, coupled to Alexa fluor 488-azide; PH, phase contrast. (C–F) UDS performed by incorporation of EdU (C and D) or BrdU (E and F) was compared. XP15BR cells are primary fibroblasts from an XP-A patient. Cells were UVC irradiated (20 J/m2), followed by incubation with 10 μM EdU or 5 μM BrdU for 2 h. Bars represent frequencies of the fluorescence levels in the indicated classes, with (black) or without (white) UVC irradiation. Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and unirradiated samples.
Mentions: We found that 20 J/m2 UV irradiation followed by 2-h EdU incubation was the optimal condition for the UDS assay. This condition was used for all experiments except for Figures 1A and 5.Figure 1.

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Show MeSH
Related in: MedlinePlus