Gretchen Lawler

DPI induces mitochondrial superoxide-mediated apoptosis

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX‐Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX‐Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX‐Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference. Cytometry Part B (Clin. Cytometry) 51B:21–29, 2003.

APPENDIX 3A Cell Counting

This unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live-dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells.

APPENDIX 2A Laboratory Safety

This appendix provides protocols for some commonly used disposal and decontamination procedures along with analytical techniques that are used to verify that reagents have been decontaminated. Some of the specific reagents covered are diaminobenzidine, ethidium bromide, cyanogen bromide and chloromethylsilane. With modification, these assays may also be used to determine the concentration of a particular chemical. Precautions are also detailed for routine handling of viable pathogenic microorganisms, as well as all human‐derived materials, because they may harbor dangerous pathogens such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and a host of bacterial pathogens.

Safe Use of Hazardous Chemicals

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.