Flow cytometry

Methodology

Procedures

One step procedure using Lysis bufer LB01, Tris-MgCl2 buffer, and Seed buffer

Two-step procedure using Otto I+II buffers


General recommendations for nuclear DNA content estimation:


Buffers and stock solutions

Otto Buffer I (Otto 1990)

0.1 M citric acid monohydrate4.2 g
0.5 % (v/v) Tween 201 ml

adjust volume to 200 ml
filter through a 0.22 µm filter; store at 4°C

Otto Buffer II

0.4M Na2HPO4 .12H2O28.65 g

adjust volume to 200 ml
filter through a 0.22 µm filter, store at room temperature
re-filter before each use

Lysis buffer LB01 (Doležel et al. 1989)

15 mM Tris363.4 mg
2 mM Na2EDTA148.9 mg
0.5 mM spermine tetrahydrochloride34.8 mg
80 mM KCl1.193 g
20 mM NaCl233.8 mg
0.1% (v/v) Triton X-100200 µl

adjust volume to 200 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter, store at -20°C in 10 ml aliquots

Tris-MgCl2 buffer (Pfosser et al. 1995)

0.2 M Tris4.84 g
4 mM MgCl2 . 6H2O162.64 mg
0.5% Triton X-1001 ml

adjust volume to 200 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter; store at 4°C

Seed buffer (Matzk et al. 2001, modified)

MgCl2 . 6H2O 0.107 g
NaCl0.5 g
Tris (Trisma-Base)1.211g
Triton X-1000.1 ml

adjust volume to 100 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter; store at 4°C

DAPI stock solution (0.1 mg/ml)

DAPI10 mg

dissolve in 100 ml H2O
filter through a 0.22 µm filter, store at -20°C in 1 ml aliquots

Propidium iodide stock solution (1 mg/ml)

Propidium iodide100 mg

dissolve in 100 ml H2O
filter through a 0.22 µm filter, store at -20°C in 1 ml aliquots

RNase stock solution (1 mg/ml)

RNase (IIA Sigma)100 mg

disolve in 100 ml H2O
filter through a 0.22 µm filter
heat to 90°C for 15 min to inactivate DNases
store at -20°C in 1 ml aliquots



References:

Doležel J., Binárová P. & Lucretti S. (1989): Analysis of nuclear DNA content in plant cells by flow cytometry. - Biologia plantarum 31: 113-120.
Matzk F., Meister A., Brutovská R. & Schubert I. (2001): Reconstruction of reproductive diversity in Hypericum preforatum L. opens novel strategies to manage apomixis. - The Plant Journal 26(3): 275-282.
Pfosser A., Amon A., Lelley T. & Heberle-Bors, E. (1985): Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. - Cytometry 21: 387-393.
Otto F. (1990): DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. - In: Crissman H.A. & Darzynkiewicz Z. (eds.): Methods in Cell Biology. Vol. 33. - Academic Press, New York. Pp. 105-110.



Plant DNA standards

Species2C DNA content (pg)*
Raphanus sativus cv. Saxa1.11 pg
Lycopersicon esculentum cv. Stupické polní tyčkové rané1.96 pg
Glycine max cv. Polanka2.50 pg
Zea mays cv. CE-7775.43 pg
Pisum sativum cv. Ctirad9.09 pg
Secale cereale cv. Dankovské16.19 pg
Vicia faba cv. Inovec26.90 pg
Allium cepa cv. Alice34.89 pg

*DNA contents estimated in Olomouc Research Centre