This protocol uses detergent-based lysis and staining solutions, which improve DNA content analysis by flow cytometry compared to staining of intact cells as in Basic Protocol 1 and Alternate Protocol 1. This protocol is suitable for tissue samples, whereas Alternate Protocol 2 provides a simplified detergent-based method designed for fixed cells or cells from suspension cultures.
Cells are collected via aspiration from tissue samples into a sucrose-citrate buffer that contains DMSO, which allows for long-term storage of samples if needed. After samples are supplemented with an internal DNA standard (a mixture of chicken and trout erythrocytes), cells are lysed, digested with trypsin, and stained with PI. The stained nuclei are then subjected to flow cytometry.
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