ROS detection questions

In measuring ROS formation in HL-60 cells and macrophages it is
recommended to use HE or 2, 7, CFDA (Dichlorodihydrofluorescein
diacetate (H2DCFDA, D399;).

1.    It appears the PMA stimulation will increase some cells greatly
(bright green). In addition to the majority of the population increases
slightly. Is this significant?

2.    Does this test measure a percentage of cells showing more activity
relative to the control (unstimulated) or is this test measuring the
amount of fluorescence increase of the major population in addition to a
limited amount of very positive green cells.

3.    How should the data be presented-linear or log scale? % bright and
mean increase of histogram?   It seems that the only histograms or
cytograms that I have seen were shown in the Methods of Cell Biology
book V42  Part B --in the Paul Robinson chapter on Functional
measurements using HL60 cells.

4.    Also Has anyone seen a difference in the improved versions of
D399?	C400 or C2938!!!

>From the Mol probes catalog--- Intracellular oxidation of H2DCF tends to
be accompanied by leakage of the product, 2',7'-dichlorofluorescein, which 
may make quantitation or detection of slow oxidation difficult. To enhance 
retention of the fluorescent product, Molecular Probes offers the 
carboxylated H2DCFDA analog (carboxy-H2DCFDA, C400), which has two negative 
charges at physiological pH, and its di(acetoxymethyl ester) (C2938), which 
should more easily pass through membranes during cell loading

bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Reproductive Toxicology Division, MD 72
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: zucker.robert@epa.gov