Castro J, Heiden T, Wang N, Tribukait B
Department of Medical Radiobiology, Karolinska Institute, Stockholm, Sweden.
Cytometry. 1993 Oct;14(7):793-804. doi: 10.1002/cyto.990140712.
An easy method for preparation of bare cell nuclei from fresh solid tissues for DNA flow cytometry is described. Pieces of up to 2 x 2 x 2 mm3 size from fresh tissues were fixed in formalin. After removal of formalin by washing with ethanol and rehydration with tap water, the tissue pieces were incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly with DAPI. Staining with ethidium bromide gave unsatisfactory results. Neither mechanical disaggregation nor centrifugation were used. The resulting cell nucleus suspensions had extremely low frequencies of debris particles and of clumped cell nuclei. A good yield, a minimized loss, and a good stainability of cell nuclei were obtained. The applicability of the method was exemplified by the analysis of biopsies from the colon-rectum in patients with ulcerative colitis and of biopsies from the bladder in patients with bladder cancer and compared to the standard method of this laboratory, which uses mechanical disaggregation, ethanol fixation, pepsin treatment, and staining with ethidium bromide. The formalin-subtilisin Carlsberg technique resulted in good agreement of ploidy measurements compared to the standard method, a higher number of evaluable histograms, an improved detectability of aneuploid cell populations, and an improved accuracy of the S- and G2-phase analysis, particularly in samples with low proliferation. The method also makes it possible to use long-term storage and to transport samples by post.
描述了一种从新鲜实体组织制备裸细胞核用于DNA流式细胞术的简便方法。将新鲜组织切成大小达2×2×2立方毫米的小块，用福尔马林固定。用乙醇洗涤去除福尔马林并经自来水复水后，将组织小块与嗜热栖热放线菌蛋白酶（链霉蛋白酶，Sigma蛋白酶XXIV）一起孵育，然后直接用DAPI染色。用溴化乙锭染色效果不理想。未使用机械解离或离心方法。所得的细胞核悬液中碎片颗粒和细胞核团块的频率极低。获得了良好的产量、最小化的损失以及细胞核良好的染色性。通过对溃疡性结肠炎患者的结肠直肠活检样本以及膀胱癌患者的膀胱活检样本进行分析，并与本实验室使用机械解离、乙醇固定、胃蛋白酶处理和溴化乙锭染色的标准方法进行比较，例证了该方法的适用性。与标准方法相比，福尔马林 - 嗜热栖热放线菌蛋白酶技术在倍性测量方面结果吻合良好，可评估的直方图数量更多，非整倍体细胞群体的可检测性提高，S期和G2期分析的准确性提高，特别是在增殖率低的样本中。该方法还使得长期保存和通过邮寄运输样本成为可能。
