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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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