Info

binding agents such as Hoechst 33342, and this effect is probably associated with ligand displacement. The high affinity of DRAQ5 for DNA is such that it will also displace ethidium bromide from DNA. A recent report has suggested that DRAQ5 may act to decrease the cellular uptake of boron dipyromethane (BODIPY)-labeled compounds (e.g., LysoTracker Green DND 26; Molecular Probes), and this has been attributed to the dyes complexing in solution (Snyder and Garon, 2003).

Health and safety

Information on DRAQ5 is available from the suppliers (see Internet Resources). Because persistent intercalating agents such as DRAQ5 have the potential to damage DNA, they should be considered both cytotoxic and potentially mutagenic. Although DNA-interacting dyes often have no defined carcinogenic potential, they should always be handled with caution.

Troubleshooting

The intense blue color of DRAQ5 in solution means that any attachment of the dye to a surface is easily detected. Adsorption to surfaces is a common problem with many dyes but is often not apparent to the naked eye. Sample tubes on flow cytometer systems operating with DRAQ5 may accumulate a blue coloration, although there is no evidence that the agent leaches into sample streams, and cross-con tamination has not been noticed. Some possible sources of problems for troubleshooting are:

1. The dye has been used in combination with an agent that may complex with DRAQ5. Check the order of addition of agents in the protocol and the appearance of the DRAQ5 solution to ensure that precipitation has not occurred. Check that the stock or any dilutions have not been frozen. It helps to have a biological control (e.g., a conveniently cultured human lymphoma suspension cell line) that can be used to check cell staining and analysis routines.

2. The sample has not been stained correctly. Check dilution calculations. At 20 |M, DRAQ5 can clearly be seen as a dark-blue solution and often imparts a blue coloration to the sample. The lipophilic properties of DRAQ5 may result in its partitioning with a reduction in the active concentration available for cell staining. For example, it is foreseeable that this may occur in samples carrying high loads of debris or vesicular matter or where cell staining has taken place in the presence of a large sink for the dye (e.g., a tissue mass, spheroids, or large clumps of cells).

3. The live sample has been held too long prior to analysis or held in a destaining buffer. Try to stain samples immediately prior to analysis, run in equilibrium with the dye, and hold cell suspensions on ice if storage for a few hours is required. Samples are usually stable >1 hr at

Figure 7.25.4 Detection of intracellular malarial parasite forms in artificially infected blood cultures using DRAQ5. (A) Fluorescence and (B) transmission images of an air-dried unfixed blood film mounted directly in PBS supplemented with 20 |iM DRAQ5. The fluorescence image shows bright regions in red blood cells, representing developmental forms of Plasmodium falciparum [NF54]. Arrows indicate an early marginal form (mf) and a later trophozoite (tr) form. Images were obtained using a Bio-Rad 1024MP confocal imaging system (647-nm excitation). Blood films were kindly supplied by Dr. Laurent Renia (Cochin, Gustave Roussy, Paris).

DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells

0 0

Post a comment