Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH<sub>i</sub>) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH<sub>i</sub>, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

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Flow cytometric measurement of proliferation-associated nuclear antigen p105 and DNA content in immuno-proliferative small intestinal disease (IPSID)

Journal of Surgical Oncology ◽

10.1002/jso.2930580106 ◽

1995 ◽

Vol 58 (1) ◽

pp. 25-30 ◽

Cited By ~ 6

Author(s):

Taner Demirer ◽

Ozden Uzunalimoglu ◽

Tom Anderson ◽

Susan M. Koethe ◽

Patrick W. McFadden ◽

...

Keyword(s):

Dna Content ◽

Intestinal Disease ◽

Nuclear Antigen ◽

Flow Cytometric ◽

Flow Cytometric Measurement ◽

Small Intestinal ◽

Small Intestinal Disease

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Intradialytic granulocyte reactive oxygen species production: a prospective, crossover trial.

Journal of the American Society of Nephrology ◽

10.1681/asn.v42178 ◽

1993 ◽

Vol 4 (2) ◽

pp. 178-186 ◽

Cited By ~ 2

Author(s):

J Himmelfarb ◽

K A Ault ◽

D Holbrook ◽

D A Leeber ◽

R M Hakim

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Fold Increase ◽

Ros Production ◽

Oxygen Species ◽

Susceptibility To Infection ◽

Flow Cytometric ◽

Ros Formation ◽

Dialysis Membranes ◽

Species Production

By the use of flow cytometric techniques, this prospective, randomized crossover study was designed to analyze intradialytic granulocyte reactive oxygen species (ROS) formation in whole blood with complement-activating and noncomplement-activating hollow fiber membranes. Dialysis with a complement-activating membrane resulted in a 6.5-fold increase in granulocyte hydrogen peroxide production 15 min after dialysis initiation and remained significantly elevated (P < 0.01) through the first 30 min with this membrane in comparison to both predialysis values and simultaneous values with a noncomplement-activating membrane. Further studies demonstrated that blood obtained at 15 min with a complement-activating membrane generated significantly less granulocyte ROS production in response to Staphylococcus aureus incubation than blood obtained either predialysis or at the same time in dialysis with a noncomplement-activating membrane. Both complement-activating and noncomplement-activating dialysis membranes caused slightly decreased granulocyte responsiveness to phorbol myristate acetate. It was concluded that hemodialysis with complement-activating membranes results in increased granulocyte ROS production and decreased responsiveness to S. aureus challenge during the dialysis procedure. These results document the potential role of ROS in hemodialysis-associated pathology and susceptibility to infection.

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STANDARDIZATION OF FLOW CYTOMETRIC MEASUREMENT OF HLA-DR MONOCYTES 53

Pediatric Research ◽

10.1203/00006450-199705000-00072 ◽

1997 ◽

Vol 41 (5) ◽

pp. 752-752

Author(s):

F Debonera ◽

A Germenis ◽

G Paterakis ◽

S Fotopoulos ◽

M Xanthou ◽

...

Keyword(s):

Flow Cytometric ◽

Hla Dr ◽

Flow Cytometric Measurement

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Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of Wilms' tumor.

Journal of Clinical Oncology ◽

10.1200/jco.1986.4.6.975 ◽

1986 ◽

Vol 4 (6) ◽

pp. 975-981 ◽

Cited By ~ 77

Author(s):

E C Douglass ◽

A T Look ◽

B Webber ◽

D Parham ◽

J A Wilimas ◽

...

Keyword(s):

Dna Content ◽

Wilms Tumor ◽

Chromosomal Rearrangements ◽

Chromosome Analysis ◽

Histologic Subtype ◽

Flow Cytometric ◽

Kaplan Meier ◽

Flow Cytometric Measurement ◽

Flow Cytometric Study ◽

Diploid Cells

Flow cytometric measurement of the DNA content of Wilms' tumor cells revealed a striking correspondence with the histologic subtype and treatment outcome. In the 48 cases studied, a hyperdiploid DNA content ranging from 1.7 to 3.2 times the result for normal diploid cells distinguished all but one of the ten anaplastic tumors. Lower values, from 1.0 to 1.4 times the diploid DNA content, characterized the nonanaplastic specimens. By Kaplan-Meier analysis, the probability of achieving 3 years of relapse-free survival was significantly lower in the group with higher DNA content (0.42 v 0.87, P less than .01). Analysis of banded chromosomes for a subset of 22 patients contributed important information beyond the flow cytometric study. Cases of anaplasia associated with poorer responses to therapy showed numerous complex translocations, whereas all others lacked such changes. By combining flow cytometric techniques and conventional methods of chromosome analysis, it should be possible to identify those patients with Wilms' tumor who are most likely to fail therapy. The biologic implication of these findings is that the development of clinical drug resistance in Wilms' tumor is a result of the genetic instability of the malignant clone.

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Electrolytic degradation of DNA fluorochromes during flow cytometric measurement of electronic cell volume.

Journal of Histochemistry & Cytochemistry ◽

10.1177/28.4.6445379 ◽

1980 ◽

Vol 28 (4) ◽

pp. 330-334 ◽

Cited By ~ 3

Author(s):

O Alabaster ◽

D L Glaubiger ◽

V T Hamilton ◽

S A Bentley ◽

S E Shackney ◽

...

Keyword(s):

Cell Volume ◽

Dna Content ◽

Chemical Degradation ◽

Quantum Yields ◽

Light Scatter ◽

Electrolytic Production ◽

Flow Cytometric ◽

Chlorine Gas ◽

Electrode Polarity ◽

Flow Cytometric Measurement

Changes in flow cytometric measurement of DNA content can result from electrolytic chemical degradation of mithramycin, ethidium bromide, and propidium iodide during simultaneous measurement of electronic cell volume. Bench electrolysis also degrades these fluorochromes without changing the quantum yields, even when they are complexed to DNA. In the flow cytometer, electrolytic production of chlorine at the anode is the probable cause of this degradation, since exposure of these fluorochromes to chlorine gas produces the same effect. It is therefore advisable to measure the DNA content distribution alone before simultaneously measuring the DNA content and the electronic cell volume. If unavoidable effects on the DNA distribution are present, narrow forward-angle light scatter should be used as the cell size indicator during dual parameter measurements. Modifying instrument design by reversing electrode polarity might eliminate this problem.

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