Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

Abstract

We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104−106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro.

Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration

“…The latter molecule is then further hydrolyzed to adenosine by other enzymes, e.g., ecto-5′-nucleotidase and alkaline phosphatase, but none of these two enzymes is present at the cell surface of resting neutrophils [13]. Alkaline phosphatase, however, is present in neutrophil granules and can be secreted as a result of neutrophil degranulation due to fMLP stimulation to subsequently generate adenosine for A 3 activation [13,19]. We know that IL-8 does not induce neutrophil degranulation unless these cells have been pre-treated with cytochalasin B to disrupt the motility response.…”

Signal transduction pathways affected by nitric oxide donors during neutrophil functional response in vitro

“…Neutrophil adherence to a plastic surface was detected by measuring the intracellular alkaline phosphatase activity of adherent cells using fl uorogenic substrate 4-methylumbelliferyl phosphate (4-MUP) [23]. Neutrophils untreated or pre-treated with 100 µM of MB, as described, were distributed into 24-well fl at bottom plate (Nunc, Denmark), (1 × 10 5 cells/well), and 10 % of FBS was added to each well.…”

Critical role of neutrophil alkaline phosphatase in the antimicrobial function of neutrophils

“…This suggests that ALP is closely related to the regression of inflammation. The activity of NAP detected by utilization of the substrate 4-MUP was associated by neutrophil adherence determination in vitro [6]. Zhang et al [21] showed that the expression levels of mNAP in patients with bacteria1 infections were significantly higher than those of patients with virus infections and the healthy controls.…”

“…Currently, what NAP acts in the biological function of the human body has not yet been clear. Utilization of the substrate 4-MUP has been recommended as a sensitive, repeatable and reliable method of neutrophil adherence determination in vitro by Bednarska et al [6]. Other studies reported that NAP localized in the secretory vesicles and organelles of neutrophils could be detected after being translocated onto the plasma membrane by f-MLP stimulation [7][8][9].…”