We present here a brief yet detailed methodology to study the activation of a number of DNA damage response factors in senescent human fibroblasts.
Method Article
Immunostaining of DDR proteins in senescent cells
https://doi.org/10.1038/nprot.2006.367
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posted 06 Dec, 2006
You are reading this latest protocol version
γ
H2AX
ATMpS1981
pS/TQ
SMC1pS996
immunofluorescence
DNA damage checkpoints
DNA damage response
senescence
oncogene-induced senescence
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)
Prepare poly-D-lysinated coverslips (poly D lysine 50μg/ml)
Plate 15-20 x 103 cells on coverslips 1 day before the staining
Wash cells gently 2x5’in PBS1x
Fix and permeabilize cells for 2’ with methanol/aceton 1:1 at RT
Block cells for 30’ with PBG1x
Stain with primary antibodies diluted in PBG1x at indicated concentration for 1h at RT in a humidified chamber
Wash cells gently 3x5’in PBG1x
Stain with secondary antibodies diluted in PBG1x for 1h at RT in a dark humidified chamber
Wash cells gently 2x5’ in PBG1x and 2x5’in PBS1x
Stain with Dapi for 5’ in the dark
Mount coverslips with 3μl of Mowiol mounting medium
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)
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 06 Dec, 2006
You are reading this latest protocol version