Enrichment of mononuclear cells from PF
Under general anesthesia, PF was collected from the pouch of Douglas at
the start of laparoscopy. Any blood-containing PF was discarded and not
included in the study. PF mononuclear cells were overlaid on the
lymphocyte separation medium (Sigma-Aldrich) and enriched by
density-gradient centrifugation at 400 rcf for 20 minutes. Cells in the
interface were harvested, washed with PBS, and incubated in 200 μl of
red blood cell lysis buffer
(ThermoFisher)
for 15 minutes at room temperature to remove any residual red blood
cells. The cells were then washed with PBS and suspended in PBS
containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) for flow
cytometry.