This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Immunostaining of DDR proteins in senescent cells
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
We present here a brief yet detailed methodology to study the activation of a number of DNA damage response factors in senescent human fibroblasts.
poly-D-lysine (SIGMA P6407 5 mg)
Methanol/Aceton 1:1
PBG 10x stock solution: 5% BSA, 2% of gelatin from cold water fish skin (SIGMA cat. G-7765) in PBS 1x
Antibodies:
γH2AX mouse Upstate Biotechnology (cat.05-636) 1:200
ATMpS1981 rabbit Rockland (cat. 600-401-400) 1:300
pS/TQ rabbit Cell Signaling Technology (cat. #2851)1:100
SMC1pS996 rabbit Bethyl Laboratories (cat. A300-050A) 1:500
DAPI (SIGMA cat.D-9564)
Mowiol mounting medium (CALBIOCHEM cat. 475904)
1) Prepare poly-D-lysinated coverslips (poly D lysine 50μg/ml)
2) Plate 15-20 x 103 cells on coverslips 1 day before the staining
3) Wash cells gently 2x5’in PBS1x
4) Fix and permeabilize cells for 2’ with methanol/aceton 1:1 at RT
5) Block cells for 30’ with PBG1x
6) Stain with primary antibodies diluted in PBG1x at indicated concentration for 1h at RT in a humidified chamber
7) Wash cells gently 3x5’in PBG1x
8) Stain with secondary antibodies diluted in PBG1x for 1h at RT in a dark humidified chamber
9) Wash cells gently 2x5’ in PBG1x and 2x5’in PBS1x
10) Stain with Dapi for 5’ in the dark
11) Mount coverslips with 3μl of Mowiol mounting medium
12) Let the coverslips dry before microscope analysis
4 h
Use always a positive (X-Rays treated cells 2-5 Gy) and a negative control (proliferating or even better contact inhibited (for a short period) quiescient cells) to reduce background
Using different type of fixative can allow better staining depending on antibody
Lipofuscin accumulation in senescent cells can give an high citosolic autofluorescent background material
d'Adda di Fagagna et al. A DNA damage checkpoint response in telomere-initiated senescence Nature 426, 194-198 (2003)
Version History
CURRENT STATUS: Posted
Version 1
Posted 06 Dec, 2006
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
Associated Publications
Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication
by Di Micco, R. et al.
Nature (03 October, 2006)
Method Article
Immunostaining of DDR proteins in senescent cells
Version History
CURRENT STATUS: Posted
Version 1
Posted 06 Dec, 2006
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
We present here a brief yet detailed methodology to study the activation of a number of DNA damage response factors in senescent human fibroblasts.
poly-D-lysine (SIGMA P6407 5 mg)
Methanol/Aceton 1:1
PBG 10x stock solution: 5% BSA, 2% of gelatin from cold water fish skin (SIGMA cat. G-7765) in PBS 1x
Antibodies:
γH2AX mouse Upstate Biotechnology (cat.05-636) 1:200
ATMpS1981 rabbit Rockland (cat. 600-401-400) 1:300
pS/TQ rabbit Cell Signaling Technology (cat. #2851)1:100
SMC1pS996 rabbit Bethyl Laboratories (cat. A300-050A) 1:500
DAPI (SIGMA cat.D-9564)
Mowiol mounting medium (CALBIOCHEM cat. 475904)
1) Prepare poly-D-lysinated coverslips (poly D lysine 50μg/ml)
2) Plate 15-20 x 103 cells on coverslips 1 day before the staining
3) Wash cells gently 2x5’in PBS1x
4) Fix and permeabilize cells for 2’ with methanol/aceton 1:1 at RT
5) Block cells for 30’ with PBG1x
6) Stain with primary antibodies diluted in PBG1x at indicated concentration for 1h at RT in a humidified chamber
7) Wash cells gently 3x5’in PBG1x
8) Stain with secondary antibodies diluted in PBG1x for 1h at RT in a dark humidified chamber
9) Wash cells gently 2x5’ in PBG1x and 2x5’in PBS1x
10) Stain with Dapi for 5’ in the dark
11) Mount coverslips with 3μl of Mowiol mounting medium
12) Let the coverslips dry before microscope analysis
4 h
Use always a positive (X-Rays treated cells 2-5 Gy) and a negative control (proliferating or even better contact inhibited (for a short period) quiescient cells) to reduce background
Using different type of fixative can allow better staining depending on antibody
Lipofuscin accumulation in senescent cells can give an high citosolic autofluorescent background material
d'Adda di Fagagna et al. A DNA damage checkpoint response in telomere-initiated senescence Nature 426, 194-198 (2003)
Associated Publications
Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication
by Di Micco, R. et al.
Nature (03 October, 2006)