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Neutrophil apoptosis was determined by flow cytometry after staining Percoll gradient-enriched leukocytes with 10 g/ml anti-CD16/CD32 mAb, 4 g/ml of APC-labeled anti-mouse CD45.2 mAb, 0.5 g/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)

FIGURE 4 According to the World Health Organization (WHO), epidemiological evidence indicates that in pregnant women, a caffeine intake of 300 mg/day (5 mg/kg day) is associated with an increased risk of intrauterine growth retardation (IUGR) ( via impacting histone acetylation and cholesterol synthesis-related gene expression via regulation of the A2AR/cAMP/PKA pathway and SIRT1 expression at the cellular level (Voerman et al., 2020)