Quantification of insulin-like growth factor I receptors on granulosa cells with flow cytometry after follicular stimulation

The presence of insulin-like growth factor I (IGF-I) receptors on granulosa cells was investigated by flow cytometric analysis. Granulosa cells were retrieved from follicular fluid after oocyte retrieval during assisted reproduction technology procedures. Whole samples of follicular fluid were pooled and the cellular fraction analysed. In order to analyse granulosa cells only we developed a dual labelling technique whereby granulosa cells were identified as CD45 negative cells, distinguishing them from leukocytes which are CD45 positive. Analysis of the IGF-I receptor was done by staining the sample with a monoclonal anti-IGF-I receptor antibody (alphaIR3 clone) and goat anti-mouse phycoerythrin labelled antibody. After identification of the presence of IGF-I receptors, receptor expression was quantified by calibration of the fluorescence signals. We analysed 10 patients' samples and showed 559-1774 binding sites per granulosa cell with a mean value of 1125 +/- 382 (SD).