Staining of DNA-synthesizing cells with BrdU-FITC
Incorporation of 5-Bromo-2-Deoxyuridine into DNA
5'??-Bromo-2'??-deoxyuridine (BrdU) is incorporated into the DNA of S-phase (DNA-synthesising) cells.
BrdU is simply added to the culture medium at a final concentration of 10-20mM together with 2'??-deoxycytidine (20-50mM). Short pulses (i.e. brief presence of BrdU in culture medium) of 10 min or less can be detected. However, for routine work pulses of 1-3 h are recommended.
Direct Immunofluorescence Staining
a) Monolayer Cells
1. Wash cells, grown on slides or cover slips, twice with PBS.
2. Fix cells with cold methanol for 20 minutes at -20C (at this stage cells may be kept for 1 month at -20C).
3. Pre-wet dry cover slips by a short incubation in PBS.
4. Denature DNA to its single-stranded form by two consecutive
treatments with 4M HCI for 15 minutes each.
5. Wash 3 times with PBS.
6. Incubate with the anti-BrdU-FITC-conjugate (30 minutes, room temperature humidity chamber).
7. Wash twice with PBS.
8. Mount dry samples with standard mounting medium and evaluate the results by fluorescence microscopy.
b) Suspension cells
1. Wash cells twice with PBS (250g, 7 minutes).
2. Resuspend cells in 3 vol PBS (0oC) and fix cells by adding 9 vol methanol (-20oC) whilst mixing the cell suspension. Incubate for 20 minutes.
3. Denature cells by adding one equal volume of 4M HCI to fixed cell suspension (20 minutes room temperature).
4. Pellet cells and resuspend the pellet in 4 M HCI, incubate for 15 minutes.
5. Carefully remove HCI by three washes with PBS (5000g, 7 minutes).
6. Resuspend cell pellet in ready to use staining solution (ca. 25ml/106 cells).
7. Wash twice with PBS.
8. Mount dry samples with standard mounting medium and evaluate the results by fluorescence microscopy.
