A) Bromodeoxyuridine/Propidium Iodide
The classical method for the analysis of cell cycle distribution is the flow cytometric measurement of DNA content which can simultaneously determine the incorporation of Bromodeoxyuridine (BrdU). The procedure requires that DNA is partially denatured to expose incorporated BrdU to a specific antibody. Denaturation is necessary because antibodies developed so far bind only to BrdU in single-strand DNA. The remaining undenatured DNA is then stained with Propidium Iodide (PI). Green fluorescence from the fluorescein-conjugated antibody is a measure of BrdU incorporation. Red fluorescence from the PI is a measure of DNA. The protocol described here uses high-molarity HCl for the denaturation of DNA. Furthermore, this method may be utilized either for unfixed or for fixed cells in suspension.

 
B) Cyclins/Propidium Iodide
Cyclins are key components of the cell cycle progression machinery. In particular, the expression of cyclins D, E, A and B1 provides new cell cycle landmarks that can be used to subdivide cell cycle into several distinct subcompartments. In this procedure cyclins expression is detectable using specific monoclonal antibodies (mAbs), and is analysed in respect to DNA content.

Generally, the peak of expression of cyclin D1 can be detected in early G1, the peak of cyclin E is typical of G1/S transition, the peak of cyclin A can be detected during G2/M phases and cyclin B1 is typical of late G2/M. Using this method, compared to the above mentioned protocol, it is possible to distinguish G0 from G1 and G2 from M phases. However, it is necessary to keep in mind that not all cell types behave in the same manner (for example, cyclin D1 is detectable not only in G0/G1 but also in G2/M, even if in a very few cell types).

 C) TUNEL/Propidium Iodide
One of the most used protocol for the determination of apoptosis in the different phases of cell cycle is the enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL). Terminal deoxynucleotidyl transferase (TdT) have been used for the incorporation of fluorescein-labeled nucleotides to DNA strands breaks in situ. DNA content is revealed by red fluorescence from PI. In order to have more details, see the Chapters related to TUNEL technique.

 D) F-Actin/Propidium Iodide
The analysis of apoptotic cells and estimation of their cell cycle specificity is also possible using a recent method. This is based on identification of apoptotic cells which have modified their cytoskleton and their DNA content. In specific, paraformaldehyde (PFA) fixation followed by staining of F-actin with fluorescein-conjugated phalloidin and of DNA with PI, are used. Furthermore, this procedure may be utilized also for adherent cells.

 
2. PROTOCOLS

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.

B.3. COMMENTARY

B.3.1 Background information
The critical steps in the methodology are cell fixation, permeabilization and the concentrations of anti-cyclin mAbs. For most cyclins optimal fixation is 70% ethanol. This treatment preserves cyclins, lowering the background, non-specific cell fluorescence and resulting in an improved signal-to-noise ratio of the cyclin specific fluorescence. Detection of D cyclin, however, requires fixation in formaldehyde. As far as anti-cyclin mAb concentration is concerned, 2.5 mg/mL is optimal for most cells. Anyway, to test the best concentration for each experimental model, is recommended.

 B.3.2 Anticipated results
In this procedure, a negative control sample, which contains only the secondary FITC-mAb, is necessary.

B.3.4 Key references

2. Faretta, M., Bergamaschi, D., Ronzoni S., D’Incalci, M., Erba, E. 1997. Diferences in cyclin B1 expression in cell cycle blocked in the G2/M phase after treatment with anti-cancer agent. A new three parametric flow cytometry analysis. Proceedings of the XIV National Italian Meeting of Cytometry.
 

3. Gong, J., Traganos, F., Darzynkiewicz, Z. 1993. Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin b measurements. Cancer Res. 53: 5096.

 
4. Gong, J., Li, X., Traganos, F., Darzynkiewicz, Z. 1994. Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle. Cell Prolif. 27: 357.
 

5. Gong, J., Traganos, F., Darzynkiewicz, Z. 1995. Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1. Exp. Cell Res. 220: 226.

 
6. Widrow, R.J., Rabinovitch, P.S., Cho, K., Laird, C.H. 1997. Separation of cells at different times within G2 and mitosis by cyclin B1 flow cytometry. Cytometry 27: 250.

C) TUNEL/PI protocol

C.2.1 Materials
formaldehyde (A2), ethanol, reaction mixture (A1), TdT buffer (A1), Bio-16-dUTP (A2), TdT enzyme (A2), staining buffer (A1), SSC buffer (A1), BLOTTO (A2), Avidin-FITC (A2), Triton X-100 (A2), PI (A2), DNAase buffer (A1).

C.3. COMMENTARY

C.3.1 Background information
This procedure is complex and not always good results are obtained. Thus, the use of commercial kits such as ApoTag^TM (Oncor, Gaithersburg, MD, USA) and "In situ cell death detection Kit" (Boeringer-Mannheim, Germany), is highly recommended.

1. Cells are fixed in 1 mL of 1% PFA for 30 min on ice.
2. Wash with 0.1% Triton X-100 in PBS, and incubate with 0.1% sodium borohydride in PBS (pH 8.0) for 30 min.
3. Wash at 200 g for 5 min.
4. Incubate with 20 mL of FITC-phalloidin (0.01-10.0 mg/mL) for 1 hour at room temperature (or overnight at 4°C).
5. As in step 3.
6. Resuspended in 1 mL of a 5-50 mg/mL PI in PBS and incubate for 30 min at 37°C.
7. Analysed with flow cytometer equipped with a 488 nm argon laser.

D.3. COMMENTARY

D.3.1 Background information
Using this protocol, the acquisition and analysis of the samples is particularly important. Apoptotic and non apoptotic cells are distinguished on the basis of the green fluorescence and the side scatter. Apoptotic cells have high side scatter and low FL-1 (1). The analysis of DNA content is relative to the different regions of apoptotic and non apoptotic cells.

D.3.3 Time considerations
The protocol is relatively simple and fast, in particular 2 hours and half are basically necessary.

 
Appendix 1: Stock solutions