Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy

Abstract

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single‐cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin‐ and type IV collagen‐coated surfaces in response to stimulation with phorbol dibutyrate or N‐formyl‐methionyl‐leucyl‐phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min. Cytometry 33:19–31, 1998. © 1998 Wiley‐Liss, Inc.

Identification of factor XIII-A as a marker of alternative macrophage activation

“…Slides were washed and mounted in an antifade containing DAPI (Vector Laboratories, Burlingame, Calif.). The relative amount of FXIII-A was determined at a single-cell level by measuring integrated fl uorescence intensity values using an Axioplan fl uorescence microscope (Zeiss, Oberkochen, Germany) connected to a black-and-white intensifi ed charge coupled device (CCD) IMAC camera (Sony, Tokyo, Japan) and a computer system (ISIS fl uorescence imaging system; Metasystems, Altlussheim, Germany) as described previously [30]. Each fl uorescence image was obtained through a × 63, NA 1.25 oil immersion objective with fi xed capture time of 0.04 s. Quantitative fl uorescent image analysis was carried out on 150-200 cells/slide.…”

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

“…After oxidation by ROS, DHR123 is converted to green fluorescent R123. 31 Both LoVo cell lines were harvested with EDTA/trypsin, then washed, centrifuged and resuspended in complete F12 medium. Cells (5 ϫ 10 5 /ml) were labeled with 1 M DHR123 for 20 min at 37°C.…”

Flow cytometric analysis of peroxidative activity in granulocytes from coronary and peripheral blood in acute myocardial ischemia and reperfusion in dogs: Protective effect of methionine

“…The strong catalytic effect of iron compounds on DHRH123 intracellular oxidation (29) suggests that hydroxyl radical, generated from H 2 O 2 by the Fenton reaction, is essentially involved in the last steps of RH123 generation, as further supported by the inhibitory effect of iron-chelating molecules (30). The importance of H 2 O 2 in DHRH123 is also stressed by the changes in DHRH123generated fluorescence in the presence of scavengers such as catalase or when the activity of H 2 O 2 -consuming enzymes is inhibited (21,23).…”

“…Intracellular generation of H 2 O 2 was estimated with the fluorochrome DHRH123 (17,(20)(21)(22)(23). Cells were resuspended (Ϸ250,000 cells/ml) in RPMI medium and incubated with 5 µM DHRH123 (from a 1 mM stock solution in DMSO) for 15 min at 37°C in the dark.…”

“…DHRH123 is taken up readily by leukocytes, and upon oxidative burst is oxidized in the presence of myeloperoxidase to yield RH123 (17). As the increase of green fluorescence is quantitatively related to the intensity of oxidative burst, the system DHRH123/RH123 has been repeatedly used for flow (17,20,21,22) and image (23) cytometry analysis of leukocyte oxidative activity. Therefore, we have applied flow cytometry to quantitate the oxidative response of granulocytes in the course of a cycle of acute coronary occlusion and reperfusion in dogs, as well as to assess the possible protective effects of methionine administration over that peroxidative activity.…”