Methods

Protocols from IPNC member labs

• Culturing of BY-2 suspension cells (PDF)
• Synchronisation of BY-2 cell culture (PDF)
• Infiltration of tobacco leaf tissue (PDF)
• Stable transformation of BY-2 cells (PDF)

Nuclei stains for live cell imaging (confocal)

Ethidium bromide

Mix 1:1 with 1x PBS, immerse plant tissue (roots, leaf sections, anthers) for 1-5mins

Excite with 514nm, sample fluorescence with 530-600nm

DRAQ5

from Biostatus

test in BY-2 cells

works best with Triton but have used with BY-2 cells without Triton

1. For BY-2's without Triton, add 1ul neat DRAQ5 to 200ul BY-2 cells and incubate for 20-40min
2. With Triton: To 500 μl cells add 1 μl 20% Triton X-100 (1 μl Triton X-100 in 5 μl dH2O) and 0.2 μl of 5 mM DRAQ5 for a working concentration of 10 μM; incubate 1-2min before imaging

Excitation with any laser from 488nm upwards; capture excitation in LP650.