A SINGLE-STEP STAINING PROCEDURE FOR THE DETECTION AND SORTING OF UNFIXED APOPTOTIC THYMOCYTES

In this study we describe a cytometric method to sort apoptotic cell f ractions, suitable for biochemical and morphological analyses. Rat thy mocytes were used as a model system, as apoptosis naturally occurs in the thymus, where the negative selection of the T cell repertoire take s place. Massive apoptosis was induced in vitro by the topoisomerase-I I inhibitor, etoposide. After etoposide treatment, a large fraction of thymocytes showed the morphological and electrophoretic features of a poptotic cells. Unfixed thymocytes were stained for 30 min with propid ium iodide (PI: 50 mug/ml containing RNase type A and detergent), and analyzed by flow cytometry. Apoptotic thymocytes typically showed sub- G1 DNA contents. Compared to non-apoptotic thymocytes, sub-G1 cells ha d lower values of low-angle (FSC) scatter and higher values of right-a ngle (SSC) scatter, so that, in dual parameter cytograms of FSC versus SSC, two distinct cell populations were apparent, and were separated by flow sorting. The purified cell fractions obtained by this procedur e had a very well preserved ultrastructural morphology; both early and late apoptotic stages (until the onset of cytoplasm segmentation) wer e present in the sorted apoptotic fraction.