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
1. Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B., Traganos, F. 1996. Cytometry of cyclin proteins.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.2.2. Methodology
1. Cells (5x10
5- 1x10
6cell/sample) are washed twice at 260 g for 5 min using 2 mL of PBS (pH 7.2).
2. Fix by 1 mL of 1% formaldehyde in PBS, on ice for 15 min.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 ApoTagTM (Oncor, Gaithersburg, MD, USA) and "In situ cell death detection Kit" (Boeringer-Mannheim, Germany), is highly recommended.
C.3.2 Anticipated results
To perform each experiment using a blank, a negative and a positive control samples, is recommended. The blank sample is assessed substituting step 9 as following: add 100
mL of reaction staining buffer prepared without Avidin-FITC. The negative sample is assessed substituting step 6 as following: add 50
mL of reaction staining buffer prepared without TdT enzyme. Blank and negative are performed in order to have a correct setting of the instrument. The positive sample, in order to make sure that the method works, is assessed by digesting with 75
mL of DNAse buffer for 20 min at room temperature before step 6.
C.3.3 Time considerations
1. Gorczyca, W., Gong, J., Darzynkiewicz, Z. 1993. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.
Cancer Res. 52: 1945.
2. Gorczyca, W.,Tuziak,T., Kram, A., Melamed, M.R., Darzynkiewicz, Z. 1994. Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labeling of fragmented DNA. Cytometry 15: 169.
3. Li, X., Darzynkiewicz, Z. 1995. Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28: 571.
D) F-Actin/PI protocol
D.2.1 Materials
PFA (A2), PBS, Triton X-100 (A2), sodium borohydride, FITC-phalloidin (A2), PI (A2).
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 flourescence 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.
In this procedure adherent cells can be utilized. In this case it is necessary to start the method as following:
ii) wash at 200 g for 5 min (continue to step 1).
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
| Solution | Preparation | Storage |
| A.Washing buffer | 0.5%Tween 20 in PBS | 4°C |
| A.Borax buffer | 0.1M Borax (Sodium tetraborate-10-hydrate) | RT |
| A. PI buffer | 3.4mM Trisodium Citrate, 9.65mM NaCl, PI 20 mg/ml, 0.03% Nonidet P-40 in H2O | 4°C |
| B.GM+EDTA buffer | glucose 1.1 g/L, NaCl 8 g/L, KCl 0.4 g/L, Na2HPO4.2H2O 0.2 g/L, KH2PO4 0.15 g/L, EDTA 0.2 g/L | 4°C |
| C.reaction mixture | 50 mL of solution was composed by: 37.8 mL of deionized water + 5 mL of TdT buffer (10X), + 5 mL of CoCl2 (25mM), + 2 mL of Bio-16-dUTP + 0.2 mL TdT enzyme | 0°C |
| C.TdT buffer (10X) | 1M Na cacodylate (pH 7.0), 1mM dithiothreitol, 0.5 mg/mL serum albumin | 4°C |
| C.staining buffer | 100 mL of solution was composed by: 54.2 mL of deionized water + SSC buffer (20X), + 20 mL of BLOTTO (25%) + 0.7 mL Avidin-FITC (160X), + 0.1 mL of Triton X-100 | 4°C |
| C.Avidin-FITC 160X | 1 mg Avidin-FITC in 250 mL PBS. Then diluit 1/10 in deionized water to have 160X stock | 4°C |
| C.SSC buffer (20X) | 0.3% sodium citrate, 3M NaCl (pH7.0) | RT |
| C.DNAase buffer | 20 ng/mL DNAasi, 10mM TRIS-HCl (pH 7.4), 10mM NaCl, 5mM MgCl2, 0.1mM CaCl2, 25mM KCl | 0°C |
Appendix 2: Reagents
| Avidin-FITC Sigma Aldrich | A2910 |
| anti-BrdU antibody Becton Dickinson | 347580 |
| Bio-16-dUTP (50nmol/50mL) Boehringer Mannheim | 1093070 |
| BLOTTO (dry non-fat milk) Bio-Rad | 170-6404 |
| Borax Riedel-Dehaen | 31457 |
| BrdU Sigma Aldrich | B5002 |
| DNAasi Boehringer Mannheim | 776785 |
| FITC-phalloidin Sigma Aldrich | P5282 |
| formaldehyde BDH | 10113 |
| goat-anti-mouse-FITC antibody Becton Dickinson | 349031 |
| Anti A cyclin antibody Santa Cruz Biotechnology | sc-239, sc-596, sc-751 |
| Anti B1 cyclin antibody Santa Cruz Biotechnology | sc-245, sc-752, sc-595, sc-594 |
| Anti D1 cyclin antibody Santa Cruz Biotechnology | sc-6281, sc-246, sc-450, sc-717, sc-753, sc-618 |
| Anti E cyclin antibody Santa Cruz Biotechnology | sc-247, sc-198, sc-481 |
| mouse serum Caltag | 10410 |
| Nonidet P-40 Sigma Aldrich | N6507 |
| paraformaldehyde Sigma Aldrich | P6148 |
| PI Sigma Aldrich | P4170 |
| Bovine serum albumin Sigma Aldrich | B7276 |
| Triton X-100 Sigma Aldrich | T9284 |
| TdT enzyme (25 U/mL) Boehringer Mannheim | 220582 |
| Tween 20 Merk-Schuchardt | 822184 |
Appendix 3: Equipment
| Flow Cabinet TC60 | Gelaire |
| Flow Cytometer FACScan | Becton Dickinson |
| Incubator CO2-AUTO-ZERO | Heraeus |
| Minifuge RF | Heraeus |
| Pipetman P20, P200, P1000 | Gilson |
| Vortex Vibrofix VF1 Electronic | Janke & Kunkel-Ika Labortechnik |
| Water Bath D8 | Haake |
首页
