**2.5 ELISA**

214 Endometriosis - Basic Concepts and Current Research Trends

concentration of macrophages in culture media was 2x106 cell/ml. Endometrial explants were cultured at 370 С and 5% СО2 for 24 hours. Samples of endometrium or peritoneal macrophages, cultured in the same conditions only in RPMI 1640, were used as controls. After termination of cultivation, endometrial tissue was washed up in phosphate-buffered saline (PBS) twice and was taken for subsequent RNA isolation or for enzymatic isolation of stromal endometrial cells*.* Peritoneal macrophages were collected from wells, filtered

The level of mRNAs expression of different factors, regulating apoptosis and invasiveness in endometrial tissue, was investigated using quantitative real time RT-PCR (reverse transcription- polymerase chain reaction). Total RNA was isolated from the whole endometrial tissue using the acid guanidinium thiocyanate-phenol-chloroform method. RNA was converted to complementary DNA (cDNA) using random hexamers and murine leukaemia virus reverse transcriptase (Promega, USA). Reverse transcription was performed at 700C for 3 min and 370C for 90 min. For real time quantitative RT-PCR, gene-specific primers and probes for human β2-microglobulin (housekeeper gene), XIAP, caspase-3, HSP27, MT-1, MMP-2, MMP-9, TIMP-2, TIMP-1 were designed and constructed in the Laboratory of Gene Engineering of National Research Center for Hematology (Moscow, Russia) using the Vector NTI Advance 10 design program (Invitrogen, USA). Commercial kit "Immunoscreen" (Gene Engineering of National Research Center for Hematology, Moscow, Russia) was used to perform real-time quantitative RT-PCR. For the thermocycle reactions and the detection of the fluorescence signals, iCycler iQ Multi-Color Real Time PCR Detection System (BIO-RAD Laboratories, California, USA) was used. To assess the number of cDNA copies in every sample, the standard curves for studied genes were constructed using control cDNA dilution series. As the controls sequences of cloned corresponding genes were used. For each endometrial sample, the amount of copies of housekeeper gene (β2-microglobulin) and specific genes were determined from the appropriate standard curve, generated by iCycler iQ software. The amount of specific gene was subsequently divided by the amount of housekeeper gene to obtain normalized specific gene value and results were presented as the ratio in a sample in the order of 103 per μl for MT-1, XIAP, caspase-3, MMP-2, TIMP-2, 104 per μl for HSP27 and 105 per μl for MMP-9 and

The surface expression of the number of functional receptors and intracellular production of proinflammatory cytokines by peritoneal macrophages was estimated with monoclonal antibodies using flow cytometry method. The following monoclonal antibodies were used in this study: FITC-conjugated anti-human-CD45, CD36, CD204, IL-1beta, IL-8, Vimentin antibodies and PE-conjugated anti-human CD14, HLA-DR, CD49e, CD11b, CD95, CD95L, IL-6, IL-12, TNF alpha antibodies (BD Biosciences, USA). Intracellular staining procedure was carried out according to the manufacturer's instructions using the FIX & PERM cell permeabilization reagents (Invinrogen, Camarillo, CA, USA). The amount of apoptotic endometrial stromal cells after incubation with peritoneal fluid or with macrophages was assessed using commercial kit with Annexin V and propidium iodide (CALTAG

through 6 layers of gauze and analyzed using flow cytometry.

**2.3 Quantitative real-time RT-PCR** 

TIMP-1.

**2.4 Flow cytometry** 

The content of IL-8, MCP-1 and calprotektin in the peritoneal fluid of women with and without endometriosis was assessed by ELISA (enzyme-linked immunosorbent assay) using commercial kits (Bender MedSystems, Austria).
