Staining Live Cells with Fluorescent Nucleic Acid Stain LUCS 13

LUCS 13 is a cell-permeant nucleic acid stain that exhibits green fluorescence upon binding to nucleic acids. The stain has a high fluorescent yield and a structure identical to SYTO™13 stain.

LUCS 13 is used to stain both DNA and RNA in live and dead eukaryotic cells as well as Gram-positive and Gram-negative bacteria. The dye is excited by the blue laser at 488 nm. Its fluorescence emission is detected in the fluorescein channel with a peak at 509 nm when bound to DNA and 514 nm when bound to RNA.

The dye can be used in simultaneous labeling with cell-impermeant nuclear markers, such as YoDi-3, to evaluate cell viability using fluorescence microscopy and flow cytometry.

Protocol

The exact protocol depends on the specific cell type and experimental task. In general, it can be described as follows:

Prepare a 50 µM stock solution of LUCS 13. To do this, add 990 µl of deionized water to 10 µl of a 5 mM LUCS 13 solution. The stock solution can be aliquoted and frozen for long-term storage.
To prepare a working 1 µM LUCS 13 solution, add 20 µl of 50 µM LUCS13 stock to 980 µl of PBS.
Carefully remove adherent cells from the growth surface in a suitable manner. With suspension cells, start work from the next point.
Wash the cells once with cold PBS (pH 7.4). Seed cells by centrifugation for 5 min at 300 g.
Incubate the cells in 1 ml of 1 μM LUCS 13 working solution for 30 min at 37°C.
Remove the dye solution by centrifugation for 5 min at 300 g.
Wash the cells once with PBS.
(Optional) If necessary, cells can be fixed in 3.7% formaldehyde in PBS for 15 min, washed with PBS, and restained with another stain.

Important

Use plastic vials when handling LUCS 13 solutions, as the diluted dye may stick to the glass.
The brighter staining results can be obtained with phosphate-free buffers (e.g.,15 mM HEPES in saline).
Before starting the experiment, test several dye concentrations ranging from 10 nM to 5 µM to determine the most optimal dye dilution. The result of staining is influenced by many factors: growth medium, cell density, presence of other cell types, duration of staining, etc.

SYTO® is a trademark of Molecular Probes Inc.