Burns E R, Bagwell C B, Hinson W G, Pipkin J L, Hudson J L
Cytometry. 1983 Sep;4(2):150-60. doi: 10.1002/cyto.990040208.
Three different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5-6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC-arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4 degrees C, with or without fixation in 10% phosphate-buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2-3% phosphate-buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.
采用三种不同的技术方案制备用于流式细胞术（FCM）分析的样本。每个开发的方案仅对某些器官效果最佳。方案I是将新鲜组织小块在碘化丙啶（PI）染色溶液中切碎，然后通过压实的玻璃棉过滤。用方案I制备的器官有：下颌下腺、膀胱、肝脏、胸腺、骨髓、脾脏、肺、肾脏和睾丸。方案II是在将器官在PI中切碎之前，先将其暴露于0.5%乙酸中48小时。用方案II制备的器官有：子宫、直肠、结肠、回肠和心脏。方案III是先将组织暴露于0.5%乙酸中，进行胃蛋白酶消化，然后用PI染色。用方案III制备的组织是角膜前表面上皮和舌表面上皮。总共成功制备了16种不同的器官和组织。对于每个器官，通过非参数和参数程序分析平均DNA直方图，并以表格形式呈现结果（时相分数）。所使用的一些器官来自暴露于1.0mg/kg长春新碱（VC）5 - 6小时的动物，以测试不同方案检测因VC阻滞有丝分裂相积累导致的G2 + M期隔室扩大的能力。通过比较在样本制备当天获得的平均DNA直方图与在4℃下储存数天至数周后同一组样本的平均DNA直方图，测试了许多不同样本制备的稳定性，样本在储存时有无用10%磷酸盐缓冲福尔马林固定。在用碘化丙啶染色后，采用最终浓度为2 - 3%的磷酸盐缓冲福尔马林固定样本的方法来确保样本稳定性。样本稳定性的证明使得样本制备可以在一个地点进行，然后将样本运输到另一个地理位置的FCM实验室。这项工作的主要发现是：a）开发了技术方案，从16种不同的小鼠器官或组织中获得了可接受的用于FCM的核悬液；b）通过用福尔马林固定PI染色的核悬液可以确保这些样本的稳定性；c）每个不同的方案都能够检测和保存至少一些被长春新碱阻滞和收集的有丝分裂相。
