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7/07
Taken from: FLUORESCENCE MICROSCOPY ANALYSIS OF NUCLEAR
Equipment
Epifluorescence microscope or Flow cytometer
(A1) Staining solution
Acridine Orange 6 g/ml
Citric acid 0.1 M
Na2HPO4 0.2 M pH 2.6
Prepare 90 ml of citric acid solution, add acridine orange and 10 ml Na2HPO4
* Acridine Orange solution is stable for several weeks when stored at 4C and in the
dark
(A2) RNAse solution
Methodology:
1. Wash cells (1x106) in PBS and centrifuge at 200 g for 5 min
2. Resuspend the cell pellet in 1 ml PBS
3. Fix cells by transferring the cell suspension in 9 ml 1% paraformaldehyde in PBS,
on ice. Incubate for 15 min on ice
4. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 5 ml PBS, centrifuge
5. Suspend the cell pellet in 1 ml PBS and transfer the suspension in 9 ml 70%
(vol/vol) ethanol, on ice.
6. Incubate for 4 h (The cells can be stored in ethanol for weeks)
7. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 1 ml PBS
8. Add 0.2 ml of RNAse A solution. Incubate at 37C for 30 min
9. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 0.2 ml PBS
10. Add 0.5 ml of 0.1 M HCl at room temperature
11. After 30-45 sec add 2 ml AO staining solution
12. Observe the cells under fluorescence microscope with an appropriate filter set.
13. Alternatively, analyse cells by flow cytometry (excitation 488 nm; dot plot of
green fluorescence at 530 20 nm versus red fluorescence >600 nm).