1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Otto I buffer in a petri dish.
1b. As an alternative, protoplasts can be prepared, and resuspended in ice-cold Otto I buffer to a concentration of 105 - 106/ml.
2. Filter the suspension through a 42 µm nylon mesh.
3. Pellet the nuclei (150g/5 min).
1. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 0.5 ml of ice-cold Otto I buffer in a petri dish.
2. Add 2 ml of Otto II buffer. It is preferable to include DAPI (or propidium iodide + RNase) in the Otto II buffer. Alternatively, these compounds can be added to the sample after the addition of Otto II buffer. The stains are used at the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Tris-MgCl2 buffer in a petri dish. It is preferable to include a DNA fluorochrome (DAPI or propidium iodid) in the buffer prior to chopping. Alternatively, this compound may be added immediately after the filtration (step 2). The stains are used in the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
In some cases, it is not possible to analyze the material immediately after collection. Then a procedure is necessary which permits storage of material for analysis at later date. This is especially critical in experiments involving the measurement of cell cycle kinetics, when samples have to be collected and analyzed at specific time intervals.
1.Mix 1 ml fresh chicken blood with 3 ml of CRBC buffer I. Centrifuge at 50g for 5 min.
2.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer I and mix gently. Centrifuge at 50g for 5 min.
3.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer II, and vortex briefly. Immediately add 2 ml of CRBC buffer III, and mix briefly.
use deionized or double-distilled water in all recipes
Tris-MgCl2 buffer (Pfosser et al. 1995)
0.2 M Tris
4.84 g
4 mM MgCl2 . 6H2O
162.64 mg
0.5% Triton X-100
1 ml
Increased sensitivity may be achieved by using a high numerical apAnalysis of DAPI Flurescence
Heat protection
KG1
Excitation filter
BG38 + UG1
Dichroic mirror
TK420
Barrier filter
GG435
Protocols for preparations of suspensions of intact nuclei sutiable for flow cytometric analysis of nuclear DNA content are described here. These protocols are routinely used in this laboratory. The procedure with LB01 buffer (Dolezel et al. 1989) works with most plants species and tissues. LB01 buffer is stored frozen at -20°C in aliquots for convenient use. For a very few species, the rezolution of DNA content histograms is not satiafactory using LB01.
It is generally agreed that flow cytometric estimation of nuclear DNA amount in absolute units should be performed using internal standard (the nuclei of a standard are isolated, stained and analysed simultaneously with the nuclei of a sample). DNA content of the sample is then calculated:
