細胞周期的流式細胞伩檢測實驗方法（PI，Brdu)2

發布時間：2019-04-27 16:34 原文鏈接：細胞周期的流式細胞伩檢測實驗方法（PI，Brdu)2

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.3.3 Time considerations
The protocol require around 2 hours before the overnight incubation and 5 hours after.

B.3.4 Key references

1. Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B., Traganos, F. 1996. Cytometry of cyclin proteins.
Cytometry. 25: 1.

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

⁶

cell/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.
3. Wash once as in step 1.
4. Resuspend in 1 mL of ice-cold ethanol 70% (at this point to store the samples at -20°C for 18 hours or overnight is possible).
5. As in step 3.
6. Resuspend in 50 mL (for each sample) of the reaction mixture, which is prepared during the last spin down.
7. Incubate for 30 min at 37°C water bath.
8. As in step 3.
9. Resuspend in 100 mL (for each sample) of the reaction staining buffer, which is prepared during the last spin down.
10. Incubate for 30 min at room temperature in the dark.
11. As in step 3.
12. Counterstain DNA with 5 mg/mL of PI in PBS.
13. Incubate for 15 min at 4 °C in the dark.
14. Analyse with flow cytometer equipped with a 488 nm argon laser.

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.

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

L of reaction staining buffer prepared without Avidin-FITC. The negative sample is assessed substituting step 6 as following: add 50

L 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

L of DNAse buffer for 20 min at room temperature before step 6.

C.3.3 Time considerations
The protocol require a quite long time. In particular 1 hour and half before the overnight incubation and a couple of hours after. Obviously, utilizing commercial kits the duration of method is highly reduced.

C.3.4 Key references

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).

D.2.2. Methodology

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:

i) add PFA 2% directly to the culture flasks for 30 min on ice. A volume equal to that in the culture flasks is added, making 1% the final PFA concentration;

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.

D.3.4 Key references
1. Endresen, P.C., Prytz, P. S., Aarbalcke J. 1995. A new flow cytometry method for discrimination of apoptoyic cells and detection of their cell cycle specificity through staining of F-Actin and DNA. Cytometry. 20: 162.

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

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