Lin P, Allison D C
Department of Surgery, Johns Hopkins Medical Institutes, Baltimore, Maryland.
J Histochem Cytochem. 1993 Sep;41(9):1435-9. doi: 10.1177/41.9.8354883.
We tested a method of measuring DNA content (Feulgen) and tritiated thymidine ([3H]-T) and bromodeoxyuridine (BrdU) incorporation by the same cell. Initial experiments showed that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. MCa-11 cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. Next, absorption cytometry was performed to measure the DNA content of randomly selected cells. Feulgen staining and the development and removal of either the [3H]-T or the BrdU grains after DNA measurements did not interfere with subsequent detection of the grains from the other label, and BrdU and [3H]-T can be used reliably in combination for identification of S-phase cells. This method may eventually allow the use of microscope-based image analysis to selectively measure the DNA contents and the BrdU/[3H]-T labeling of non-transformed stromal and cancer cells in solid tumors, thereby providing new insights into the growth kinetics of these heterogeneous cell populations.
我们测试了一种通过同一细胞测量DNA含量（福尔根染色法）以及氚标记胸腺嘧啶核苷（[3H]-T）和溴脱氧尿苷（BrdU）掺入量的方法。初步实验表明，福尔根水解能充分使固定细胞的DNA变性，以便用单克隆抗体检测掺入的BrdU。然后用[3H]-T和BrdU对MCa-11细胞进行双重标记，置于载玻片上，进行福尔根染色。接下来，进行吸收细胞计数以测量随机选择细胞的DNA含量。福尔根染色以及在DNA测量后[3H]-T或BrdU颗粒的显影和去除均不影响随后对另一种标记颗粒的检测，并且BrdU和[3H]-T可可靠地联合用于鉴定S期细胞。该方法最终可能允许使用基于显微镜的图像分析来选择性地测量实体瘤中未转化的基质细胞和癌细胞的DNA含量以及BrdU/[3H]-T标记，从而为这些异质性细胞群体的生长动力学提供新的见解。
