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After removing the medium, the cells were washed twice with icecold phosphatebuffered saline (PBS) and harvested with 1 ml icecold PBS containing a 1 protease inhibitor cocktail (P3100005, Gendepot) followed by centrifugation at 3,000 g at 4C for 5 min

doi:10.1186/s12882-018-1101-4

Cells were fixed in 4% PFA and stained with DAPI and imaged with a ZEISS LSM 900 confocal microscope

Study limitations Although pomegranate extract effects on TMAO production were observed in female participants of premenopausal age and male participants, this remains a proof-of-concept study with a small population and smaller subgroups, including only three female participants in the Older (postmenopausal age) group and three in the Younger (premenopausal age) group