DNA synthesis assay with 3H-TdR doping assay

Summary

Cells were labeled with 3H -TdR and DNA was extracted to determine radioactivity levels by scintigraphy Source : Animal Cell Culture : A Guide to Basic Techniques 5th Edition

Operation method

Scheme 21.5 DNA synthesis assay with 3H-TdR doping assay

Principle

Cells were labeled with 3H -TdR and DNA was extracted to determine radioactivity levels by scintigraphy

Move

makings

non-sterile
Cells at the appropriate time period (if using the hot 2mol/L PCA method requires cells to be grown in glassware or test tubes, if using other methods, cells can be cultured in regular plastic dishes)

^3H-TdR, 0.4 MBq /ml (~10uC i/ml), lOOul per ml of culture medium

Some media, such as Ham's FlO and F12, contain thymidine deoxyriboside. It will ultimately determine the specific activity of the added ^3H -TdR. It is therefore important to take this factor into account when determining the amount of isotope to add. While an isotope of 40KBq/ml (~1.0Ci/m l) is sufficient for most media, 0.2MBq/ml (~5uCi/ml) should be used for F10 or F12.

Non-sterile
Trichloroacetic acid (TCA ), 0.6 mol/L (on ice), 6 ml per ml of culture medium.
HBSS or D-PBSA, on ice, 2 ml per ml culture solution
Gallic acid, 2 mol/L or 0.3 m o l/L NaCl containing 35 mmol/L sodium dodecyl sulfate (SLS, SDS), 0.5 ml per ml of medium
MeOH, lml per ml of medium
Liquid Flash Bottles
Scintillation solution (10X perchloric acid or NaOH/SDS)

Procedure

1. Cells are cultured to the desired density (usually the highest DNA synthesis is in the mid-log phase or the plateau phase of density-limited DNA synthesis, (see Section 18.5.2).

2. Add ^3H -TdR dissolved in 40KBq/ml (~1.0Ci/ml) or 2MBq/mmol (~50Ci/mol) of HBSS.

3. Incubate the cells for 1~24 h as required for the experiment.

4. Carefully aspirate the radioactive culture solution and place it in an appropriate container of liquid radioactive waste.

5. Carefully wash the cells with 2 ml of HBSS or PBSA, add 0.6 mol/L 2 ml of pre-cooled TCA on ice, and allow to stand for lOmin. If the cells are loosely adherent, fix the cells with MeOH (see Scheme 21.6).

6. Wash the cells twice with trichloroacetic acid for 5 min each time.

7 . Add 0.5 m1 2mol/L perchloric acid and place on a hot plate at 60°C for 30 min, then allow to cool. Alternatively, add 0.5 ml of SLS dissolved in NaOH and incubate at 37°C for 30 min or overnight at room temperature.

8 . The soluble precipitate is collected and transferred to a scintillation solution and the radioactivity is measured in a scintillator.

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