Labeling index experiments with 3H-thymine deoxyriboside
Labeling index experiments with 3H-thymine deoxyriboside
Cells are cultured to the appropriate density, labeled with 1H -thymidine deoxyriboside for 30 min, washed and fixed, and unadulterated precursors are removed in preparation for isotope radiation autoradiography. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 21.11 Labeling index experiment for 3H-thymine deoxyriboside
Principle
Cells were cultured to appropriate density, labeled with 1H -thymidine deoxyriboside for 30 min, washed and fixed to remove unadulterated precursors, and prepared for isotope radioautography. Move makings For more product details, please visit Aladdin Scientific website.
non-sterile
Cultured Cells
Growth solution
0.25% crude trypsin
D-PBSA
3H-thymidine deoxyriboside, 2.0 MBq/ml (~50uCi/ml), 75 GBq/mmol (~2Ci/mmol)
Caution should be exercised when handling 3H-thymidine deoxyriboside as it is a radioactive substance and a gene product! Follow local guidelines (see section 7.7).
Multiwell plate with 13 mm Thermanox cover slip (Nalge Nunc)
Non-sterile
Hematocrit plate or electronic counter
D-PBSA
Methanol acetate (1 part glacial acetic acid to 3 parts methanol), ice cold, freshly prepared
Microscope slide
Sealer (e.g. DPX or Permount)
Trichloroacetic acid (TCA ), 0.6mol/L, ice-cold
Deionized water
Methanol
Procedure
1. In a 24-well plate with a cover slip, culture the cells at 2X104-5X104 cells/ml.
2. Allow the cells to adhere to the wall and begin to proliferate (48-72 h) to reach the desired cell density.
3. Add 3H-thymidine deoxyriboside, 100KBq/ml (about 5uCi/ml) to the culture medium and continue to incubate the cells for 30 min.
Some media, such as Ham's F10 and F12, which contain thymidine deoxyriboside (Tables 9.3, 10.1 and 10.2), require that the concentration of radioactive thymidine deoxyriboside be increased so that the same specific activity is achieved within the culture medium.
4. Remove the labeled liquid and dispose of it in a dedicated radioactive waste container.
5. Wash the coverslips three times with D-PBSA. With each rinse, lift the capsule from the bottom of the hole (but not beyond the hole) so that the isotope at the bottom of the capsule can be washed out.
6. Add 1:1 D-PBSA:methanol acetate, lml per well, and aspirate immediately.
7. Add lml of glacial acetic acid (4°C) to each well and let stand for l0 min.
8. Remove the cover slip and blow dry with a fan.
9. Place the cover slip on a microscope slide with the cell side facing up.
10. Allow the sealer to dry overnight.
11. Place the slide in a staining vat (4°C) containing 0.6mol/L TCA for lO min, replacing the TCA twice during the process to remove unadulterated precursors.
12. Rinse with deionized water, then change to methanol and air dry.
13. Prepare for radioautoradiography (see protocol 27.3.).
14. When the autoradiograph has been exposed for a period of time (usually 1 to 2 weeks), it is developed, stained (see Scheme 27.3), and visualized under a 40× microscope.
15. Count the percentage of labeled cells. To cover a representative area, scanning observations are made as shown in Figure 21.14.
