1. Cells (1x10⁶/mL) are incubated with BrdU 10 mM at final concentration, for 30 min at 37 °C in controlled atmosphere.
2.Wash twice at 500 g for 1 min using the washing buffer.
3. Resuspend in 0.5 mL of washing buffer and 0.5 mL of HCl 4 M.
4. Mix accurately and incubate for 30 min at room temperature.
5. Wash once as in step 2.
6. Resuspend in 1 mL of Borax buffer.
7. As in step 5.
8. Resuspend in 200 mL of washing buffer and label with 5 mL of mAb ant-BrdU.
9. Incubate for 1 hour at 4 °C in the dark.
10. As in step 5.
11. Resuspend in 200 mL of washing buffer and label with 4 mL of goat-anti-mouse FITC-conjugated antibody.
12. Incubate for 30 min at 4 °C in the dark.
13. As in step 5.
14. Resuspend in 200 mL of washing buffer and 200 mL of PI buffer.
15. Incubate for 15-30 min at 4 °C in the dark.
16. Analyse with flow cytometer equipped with a 488 nm argon laser.

A.3. COMMENTARY

A.3.1 Background information
In this procedure fixed cells by 4% PFA in Phosphate Buffer Saline (PBS) can be utilized. In this case to wash cells once in PBS before to start at step 1 is necessary.

Moreover, both direct and indirect immunofluorescence can be used. The BrdU incorporation is more evident using the indirect method.

B) Cyclins/PI protocol

B.2.1 Materials
PFA (A2), Triton X-100 (A2), PBS, mouse serum (A2), mAbs anti-cyclins (A2), goat-anti-mouse-FITC (A2), PI (A2), GM+EDTA buffer (A1).

In the case of E, A and B1 cyclins, cells (2x10⁶/mL) are fixed by 70% ethanol:

a) put all the reagents in ice;
b) count and centrifuge cells at 375 g for 5 min;
c) resuspend accurately the pellet in 1mL GM+EDTA buffer ;
d) add gently 3 mL of 96% ethanol vortexing samples and working in ice;
e) store the fixed samples at 4°C.
 

In the case of D cyclins, cells are fixed by 1% methanol-free formaldehyde:

a) put all the reagents in ice;
b) count and centrifuge cells at 375 g for 5 min.;
c) resuspend gently the pellet in 1 mL of 1% formaldheyde in PBS for 15 min in ice;
d) wash in ice-cold PBS as in step b;
e) add 1 mL of 70% ethanol;
f) store the fixed samples at 4°C.

1. Wash with ice-cold PBS.

2. Resuspend the pellet in 1 mL of 0.25% Triton X-100 in PBS, vortexing samples and working in ice (for 5 min in the case of D, A and B1 cyclins, for 10-20 min in the case of E cyclin).

3. Wash in ice-cold PBS as in step b.

4. Incubate cells with 150 mL of mAb anti-cyclin (diluited at the concentration of 2.5 mg/mL in PBS + 1% of normal goat serum) overnight at 4°C.

5. As in step 3.

6. Incubate for 1 hour with 150 mL secondary mAb goat-anti-mouse-FITC (1.3 mg/mL) diluited 1:50 in PBS + 1% normal goat serum.

7. As in step 3.

8. Incubate for 4 hours at room temperature and in the dark with 1 mL of 2.5 mg/mL PI in PBS (+ 12.5 mL of 1 mg/mL RNAsi) or incubate overnight at 4°C with 0.8 mg/mL PI in PBS (+ 12.5 mL of 1 mg/mL RNAsi).

9. Analyse with flow cytometer equipped with a 488 nm argon laser.