Abstract |
We have developed an improved method for measuring the filamentous ( F) actin content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleton. Fresh whole blood (100 microliters) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer pre-cooled to -8 degrees C and subsequently fixed at 4 degrees C for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugation, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothiocyanate ( FITC)- phalloidin and quantified by means of flow cytometry. In healthy test subjects, PMN stimulation by the chemotactic peptide formyl- methionyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly increased F-actin assembly, while in patients with multiple organ failure, two subpopulations arose: one with greater F-actin content and a second with lower F-actin content in comparison with the unstimulated blood sample. This simple and fast method may be a useful tool in basic and clinical research.
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Authors | G Egger, A Burda, A Glasner |
Journal | Virchows Archiv : an international journal of pathology
(Virchows Arch)
Vol. 438
Issue 4
Pg. 394-7
(Apr 2001)
ISSN: 0945-6317 [Print] Germany |
PMID | 11355175
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Actins
- Phalloidine
- N-Formylmethionine Leucyl-Phenylalanine
- Fluorescein-5-isothiocyanate
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Topics |
- Actins
(blood)
- Adult
- Cytoskeleton
(chemistry)
- Female
- Flow Cytometry
(methods)
- Fluorescein-5-isothiocyanate
(chemistry)
- Humans
- Male
- Multiple Organ Failure
(blood)
- N-Formylmethionine Leucyl-Phenylalanine
(pharmacology)
- Neutrophils
(chemistry, drug effects)
- Phalloidine
(chemistry)
- Reference Values
- Staining and Labeling
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