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Neutrophil apoptosis was determined by flow cytometry after staining Percoll gradient-enriched leukocytes with 10 μg/ml anti-CD16/CD32 mAb, 4 μg/ml of APC-labeled anti-mouse CD45.2 mAb, 0.5 μg/ml of Brilliant violet 421-labeled anti-mouse Ly6G, as well as Alexa Flour 488-labeled annexin V and propidium iodide (PI) using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit (Invitrogen)

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Furthermore, the measurement of cytokines was a methodologically challenging endeavor at that time because of the absence of sensitive assays