Combined Thymidine Autoradiography and Cell Staining for Senescence-

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Tony Rook
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Combined Thymidine Autoradiography and Cell Staining for Senescence-

Please find the following protocol:

Combined Thymidine Autoradiography and Cell Staining for Senescence-Associated β-Galactosidase (SA-β-Gal) Activity

Protocol:

Radiolabeling With [3H]-Thymidine

1. Incorporate [3H]-Thymidine label into cells by adding 10 μCi/ml [3H]-Thymidine to the growth media.

2. Incubate for between 48 to 72 hours.

Β-Galactosidase Staining

1. Carefully remove the growth media from the cell culture.

2. Add an equal volume of PBS and gently mix the PBS solution around to wash the cells of the cell culture media.

3. Repeat the PBS wash of the cells two more times.

4. Discard the PBS wash solution.

5. Add Fixative Solution to the cells and incubate at room temperature for 3 to 5 min. Cells can also be fixed in 3% (v/v) Formaldehyde in PBS; however the Fixative Solution preserves morphology better.

6. Wash cells as before in PBS two times.

7. Add between 1 to 2 ml of Staining Solution per 35 mm culture dish or cover slide.

8. Incubate for at least 2 hr at 37°C (do not incubate in a CO2 incubator). A blue color is visible within 2 hr; however, staining is maximum at between 12 to 16 hr. Blue stain is indicative of senescence-associated β-galactosidase activity.

Cell Fixing for Autoradiography of [3H]-Thymidine

1. Wash cells as in Step#2 with PBS two times.

2. Wash cells with Methanol two times.

3. Allow cells to air dry.

4. Coat cells with NTB2 Emulsion and incubate in the dark at room temperature for 12 to 48 hours.

5. Develop using Microdol X (follow manufacturer's instructions).

6. Fix cells in Rapid-Fix (Kodak).

7. Rinse cells with a copious amount of ddH2O.

8. Autoradiograph the cells and compare [3H]-Thymidine incorporation with senescence-associated β-galactosidase activity.

Solutions:

X-Gal Solution in DMF (CAUTION Biohazard!)
20 mg/ml X-Gal
(5-bromo-4-chloro-3-indoly-Β-D-galactopyranoside)
Store -20°C
X-Gal is not stable in water
Should be added to the Staining Solution just before use

100 mM Potassium Ferricyanide
100 mM Potassium Ferricyanide (K3Fe(CN)6)
CAUTION Biohazard!

100 mM Potassium Ferrocyanide
100 mM Potassium Ferrocyanide (K4Fe(CN)6)
CAUTION Biohazard

Citric Acid/Phosphate Buffer
0.1 M Citric Acid
0.2 M Sodium Phosphate Dibasic (Na2HPO4)
Proper pH is VERY important
pH 6.0

PBS
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl

[3H]-Thymidine in Cell Culture Media
10 μCi/ml [3H]-Thymidine (50 to 80 Ci/mmol)
CAUTION Biohazard

Staining Solution
1 ml of 100 mM K3Fe(CN)6 (final concentration 5 mM)
40 μl of 1 M MgCl2 (final concentration 2 mM)
*Add just before use
600 μl of 5 M NaCl (final concentration 150 mM)
Prepare Staining Solution the day of experiment
4 ml of Citric Acid/Phosphate Buffer
(final concentration 30 mM)
1 ml of 100 mM K4Fe(CN)6 (final concentration 5 mM)
1 ml of X-Gal Solution* (final concentration 1 mg/ml)

Cell Fixative in PBS
2% (v/v) Formaldehyde
0.2% (w/v) Glutaraldehyde

1 M MgCl2

5 M NaCl

Bioreagents and Chemicals:

Potassium Ferricyanide (III)
Sodium Phosphate, Dibasic
Glutaraldehyde
Potassium Chloride
Sodium Chloride
Methanol
Formaldehyde
Potassium Phosphate, Monobasic
[3H]-Thymidine
Magnesium Chloride
Rapid-Fix
Microdol X
NTB-2 Emulsion
DMF
X-Gal
Citric Acid
Potassium Ferrocyanide (II)

References and Links:

1. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et. al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U S A. 1995 Sep 26;92(20):9363-7.

http://www.bio.com/protocolstools/protocol.jhtml?id=p9025