A sequential methodology that allows apoptotic cell sorting and FISH analysis in human testicular cells

Abstract

The objective of this study was to develop a methodology that permits the detection and separation of apoptotic cells in human testicular tissue and their subsequent cytogenetic analysis by fluorescence in situ hybridization (FISH). The sequential methodology consisted of five steps: 1) enzymatic disaggregation of testicular tissue, 2) specific staining of apoptotic cells, 3) cell sorting by flow cytometry, 4) cell fixation, and 5) FISH. Enzymatic disaggregation yielded cell counts that ranged from 1.7x105 to 5x106 cells, and viability values greater than 72%. The apoptotic (mean ± SD: 22% ± 5.3%) and viable (45.5% ± 7.3%) populations were identified and selected by flow cytometry and demonstrated purity values ranging between 62% and 100%. The paraformaldehyde fixation of the selected fractions resulted in cell loss values of less than 10%. The application of three treatments before FISH (membrane permeabilization, elimination of cytoplasmic components, and re-fixation of the sample) resulted in hybridization frequencies of greater than 98%. In both selected fractions, cells of all spermatogenic stages and Sertoli cells were identified. The methodology developed has enabled the preparation of a cellular suspension with optimal viability and counting, the efficient selection of the apoptotic population, and its analysis by cytogenetic techniques. The application of this methodology in testicular cells should help establish whether there is a direct relationship between chromosome anomalies and apoptosis.