HI,

You can use acridine orange along with Ethidium bromide. Acridine orange alone wont stain the apoptotic cells. If apoptotic cells are there ethidium bromide enters the cell because of permeability of the cells and binds to the DNA.

Jagan

Acridine orange is a complex dye to work with. It is cell permeant so it can penetrate living as well as dead cells. The change in wavelength depends on binding to either single-stranded or double-stranded nucleic acids and as such has been used to measure both DNA and RNA (single-stranded) in cells. The color is green when it binds to ds-DNA (or ds-RNA) and red when it binds to ss-RNA (or ss-DNA). The binding to DNA depends on the stoichiometry of the AO vs the DNA or RNA and it can lead to misinterpretation if it is not carefully controlled. Phagocytosed cells could be red either because of ss-RNA or degraded DNA that has ss regions. For these reasons it is a complicated dye to use and probably explains why more people haven't used it for the application you propose.

Besides binding to both RNA and DNA, it is a biohazardous dye. Unless you make the right dilution it can cause you a lot of trouble. We use AO for measuring the DNA breaks in sperm and it works very well.

A dye to use for phagocytosis is DHR123 (Dihydrorhodamine123) which becomes fluorescent with mitochondria activity. You may use different stimulants (bacteria, C3b, PMA, fMLP) and obtain data on most of neutrophil functions. If you look up Molecular Probes Book on Invitrogen web site, you will see that there are many different dyes serving the purpose without causing any biohazard.

How do you use DHR123 to monitor phagocytosis? I thought it' a probe used tu detect ROS.

We isolate the granulocytes by density gradient. Then stimulate with PMA for oxidative burst, with BACTERIA for PHAGOCYTOSIS and with fMLP for chemotaxis. We stain the cells with RHD123, and make measurements at zero and at 20 minutes (we have found that 20 minutes is the optimum time period to get highest fluorescence). If the cells are stimulated then RHD123 fluoresces (due to mitochondria activation) and we calculate the index value from the mean/median numbers obtained with unstimulated cells. Bacteria we use are St. aereus and E. coli (both are obtained from ATCC but do not have the cat. numbers at the moment). We always run a healthy control sample with these measurements. Our method is improvised over a method developed by G. Rothe et al. which was published years back in Methods in Molecular Biology series on flow cytometry.

Hi all,

First thank you very much for your answers. AO seems not be a reliable dye for observing phagocytosed cells.

I'll have a look to the Molecular Probe book.

But I already found a dye which seems interesting: CypHer 5. It fluoresces in ph-dependent manner. Did anyone use it?

Best