A Simple Method for Cell Isolation from Paraffin-Embedded Tissue Spscimens for Flow Cyrometric DNA Analysis.

Creators Oga Atsunori

Creators Ohzono Yuko

Creators Yoshizawa Hiroto

Creators Sasaki Kohsuke

Since Hedley et al. developed a cell isolation method using pepsin from a paraffin-embedded material, may archival tissues have been analyzed by flow cytometry. The original and modified methods have greatly contributed to DNA ploidy and cell cycle onalyses, in particular, to retrospective studies. However, the methods need repetitive centrifugations and/or long incubation time. For saving time and efforts we aimed to improve the Hedley's method. Tissues used ware normal lymph nodes and malignant lymphonoma of testis. All cell isolation procedures were done in a 1.5-ml microfuge tube without centrifugation. Tissue sections ware mined with scissors, deparaffinized and rehydrated. Subsequently, the tissue was treated with 0.1% pepsin in 0.1N HCl for 90 min at 37℃ and neutralized. After filtering out cell debris, the cell suspension was treated with 0.1% Rnase and stained with propidium iodide. The average coefficient of variation for G0/G1 peak of DNA diploid cells was 2.6%, and it was small enough to detect a near diploid DNA aneuploid peak (DNA index: 1.13). All procedures can be completed within 4 hours without difficulty. This method is suitable for lymphocytic tissues.

[ISSN]0513-1812

[NCID]AA00594272