*4.6. NK Cell Cytotoxicity Assay*

PBMC were isolated from the buffy coat by Histopaque®-1077 (Sigma-Aldrich) density gradient centrifugation, washed and cultured in RPMI 1640 + GlutaMAX medium supplemented with 10% FBS and 1% antibiotic–antimycotic solution (all from Invitrogen, TermoFisher Scientific) with or without addition of PF (1:1) from patients with endometriosis or control subjects at a density of 2 <sup>×</sup> <sup>10</sup>6/mL in 12-well plates at 37 ◦C in 5% CO<sup>2</sup> atmosphere. Following 24 h of culture natural cytotoxic activity of PBMC was evaluated by means of NKTEST™ (Glycotope Biotechnology, Heidelberg, Germany) according to the detailed description provided by the manufacturer. In brief, cultured effector PBMC and K562 target cells prestained with a green fluorescent membrane dye were mixed at 50:1, 25:1 and 12.5:1 effector-to-target (E:T) ratio in a test medium and incubated for 3 h at 37 ◦C in 5% CO<sup>2</sup> atmosphere. Following incubation, the cells were stained with DNA staining solution for 5 min at 4 ◦C and the cytotoxicity was measured using CytoFLEX (Beckman Coulter) and CytExpert 2.0 software. Specific cytotoxicity was calculated on the basis of analysis of 5000 target cells and shown as percentage of positively stained cells. The results of cytotoxicity of PF-preincubated effector cells were presented in relation to control effector cells preincubated in medium alone.
