One small section was washed in phosphate buffer saline and fixed in ice cold neutral buffered paraformaldehyde, pH 7. 4, for histological assessment, and the promotion re sidual tissue was processed for tissue dissociation and cell separation as described below. Cell isolation and separation Enzymatic dissociation of endometrial Inhibitors,Modulators,Libraries tissue followed by cell separation was performed using previously described standardised procedures. Briefly, the endomet rial specimen was washed in HBSS, minced and incubated at 37 C with collagenase III in HBSS supple mented with 2% foetal calf serum, 15 Inhibitors,Modulators,Libraries mM HEPES, 1X antibiotic antimycotic solution and gentamicin for 30 minutes with shaking at 50 rpm. The final cell suspension contained primarily single stromal cells and fragments of Inhibitors,Modulators,Libraries glands.

The cell suspension was washed three times with Ca Mg free HBSS supplemented with the antibiotic antimycotic mixture and filtered through a sterile, pre equilibrated mesh filter. The filtrate contained the stromal cell enriched fraction. The residual epithelial cell enriched fraction was further purified by Inhibitors,Modulators,Libraries unit gravity sedimentation in 10% FBS. The stromal cells remained in cell suspension, and the sediment, consisting of sheets, fragments and clumps of epithe lial cells, was collected and washed three times with HBSS mod. The epithelial cell fraction was further enriched by density dependent fractionation on a dis continuous Percoll gradient as described previously. The epithelial cell enriched fraction was re suspended in complete medium and stored on ice.

The stromal cell enriched fraction was further enriched by density dependent fractionation on a discontinuous Percoll gradient as described previously. CD45 positive leucocytes were depleted using anti CD45 MACS microbeads and LS magnetic separation columns in a MidiMACS separator as described. The negative fraction con taining the stromal cell enriched population was washed Inhibitors,Modulators,Libraries twice with HBSS mod at 300 g for 10 minutes and re suspended in 1 ml of complete medium and kept on ice. For separated mixed cells, endo metrial cells separated by enzymatic digestion were not passed through a mesh filter, but the remaining steps were followed as sellckchem described above. The yield and viability of the isolated cells were determined using standard proto cols. The relative abundance of cytokeratin positive, vimentin positive, CD45 positive and vW factor positive cells in all three groups was determined using standard immunocytochemical procedures. Primary culture of human endometrial cells Primary cell cultures at 1×105 cellscm2 were separated into three different groups Group 1 epithelial cells, Group 2 stromal cells, and Group 3 mixed cells.