Percoll公司
卵泡液
流式细胞术
颗粒细胞
生物
细胞
细胞生物学
体外
化学
分子生物学
卵泡期
内分泌学
卵母细胞
胚胎
生物化学
作者
Michael C. Quinn,SheilaJ Mcgregor,Jo‐Ann L. Stanton,Paul A. Hessian,Wayne R. Gillett,D. P. L. Green
摘要
Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.
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