*4.2. RNA and Protein Isolation in Peritoneal Fluid-Treated Cells*

Peritoneal fluid (PF) (devoid of blood contamination) was collected on ice from all women during laparoscopic surgery. Peritoneal fluid was spun at 2000× *g* to remove any cellular debris. The supernatant was used immediately for studies or stored in a −80 ◦C freezer for future use. To establish a cell model of the peritoneal environment, Ishikawa cells, a human (39-year-old woman) established endometrial epithelial cell line (Cat No: 99040201, Sigma-Aldrich, St. Louis, MO, USA), were cultured in T75 flasks in complete media (DMEM/F12, 10% FBS, 1% Pen/Strep, 1% L-glutamine). These cells were used because they express characteristics similar to those of mature endometrial epithelial cells [74–76]. Approximately 70% confluent cells were treated with 1% PF from patients for 48 h in a DMEM/F12 media containing 1% charcoal-stripped FBS. Patient peritoneal fluid (PF) groups were control PF (fluid from women without endometriosis) and endo PF (fluid from women with endometriosis). The concentrations of PF chosen were based on our previous published studies [33,55]. At the end of the 48-h treatment, cells were collected using Qiazol Lysis reagent (Cat No: 79306, Qiagen, Gaithersburg, MD, USA) and RNA was isolated using the Qiagen miRNeasy Mini Kit (Cat No: 217004, Qiagen, Gaithersburg, MD, USA). The quantity and quality of RNA were measured in the NanoDrop 2000 spectrophotometer. Cell lysates for measuring proteins, were prepared in RIPA buffer containing protease inhibitors (Cat No: P2714, Sigma-Aldrich, St. Louis, MO, USA) and protein concentrations were measured using a modified Lowry protocol [77].
