Oxidative burst assessment and neutrophil-platelet complexes in unlysed whole blood.
BACKGROUND
Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation.


METHODS
To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated.


RESULTS
With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not.


CONCLUSIONS
This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.
Avendaño
Ariadna
A
Institut de Recerca de l'Hospital Universitari Vall d'Hebron, Barcelona, Spain.
Sales-Pardo
Irene
I
Marin
Lorena
L
Marin
Pedro
P
Petriz
Jordi
J
D000880:Anthraquinones / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D001792:Blood Platelets / Q000276:immunology* / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects / Q000276:immunology*; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones; D000911:Antibodies, Monoclonal; D002273:Carcinogens; D007476:Ionophores; D018950:Lipopolysaccharide Receptors; D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015054:Zymosan; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D013755:Tetradecanoylphorbol Acetate