Hello Verena Kast

You may use the myeloperoxidase (MPO) activity assay as a measure of functionality for human neutrophils. MPO, the most abundant protein in neutrophils plays a major role in many inflammatory pathologies. This active enzyme is mainly stored within cytoplasmic azurophilic granules in the neutrophil until cell activation.

Upon neutrophil activation, primary granules containing MPO can fuse with the plasma membrane to secrete contents into the extracellular milieu. During respiratory burst, MPO utilizes H2O2 to catalyze the formation of ROS, including HOCl, hypothiocyanous acid (HOSCN), hypobromous acid (HOBr) and hypoiodous acid (HOI) that reacts with proteins, unsaturated fatty acids and any oxidizable group, to induce protein adducts and genetic mutations, and influence signaling pathways. It is suggested that extended exposure to ROS due to chronic inflammation results in DNA damage and genomic instability leading to malignant cell transformation. You may want to refer to the paper attached below.

Article Myeloperoxidase: Growing importance in cancer pathogenesis a...

For measurement of MPO release, human neutrophils suspended in HBSS at 4 x 10^6 cells/ml in a 15-ml tube may be stimulated with 25-100 nM phorbol 12-myristate 13-acetate (PMA) for up to 2.5 - 3 hours. The reaction may be terminated by placing the tube on ice and centrifuging at 400g for 5 min at 4-degree C. MPO activity in the cell supernatant may be assayed using tetramethylbenzidine (TMB).

You may add 100µl of TMB substrate solution to 50 µl of cell supernatant in a 96-well microplate followed by incubation at room temperature for 15 min. The reaction may be terminated by the addition of 50 µl of 1M H2SO4, and absorbance may be read at 450 nm in a spectrophotometric microplate reader.

Best.

Thank you very much. This is really helpful.

As a generality mature neutrophils don't survive freezing well. On thawing you are likely to have lysed neutrophils. Thus, not sure this would be a viable comparison.

You might find the paper An easy and reliable whole blood freezing method for flowcytometry immuno-phenotyping and functional analyses of interest. The authors report that following freezing 20% of the neutrophils are lost on thawing (presumably lysed) and of the remaining neutrophils 42% were dead/dying. "FSC low PMs were CD15 + CD16+ live/dead dim dying neutrophils (mean frequency: 42% of total PMs) (Figure 2(b)).

Thank you very much James.

We isolate neutrophils by density gradient separation and commonly use mieloperoxidase (MPO) activity assay to measeures funcionality.

Generaly isolated neutrophils cultured for 24 hrs are functional and good quality to use in others experiments.