Lan H Y, Hutchinson P, Tesch G H, Mu W, Atkins R C
Department of Nephrology, Monash Medical Centre, Vic, Australia.
J Immunol Methods. 1996 Mar 28;190(1):1-10. doi: 10.1016/0022-1759(95)00233-2.
Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens. Thirdly, microwave treatment also produces a discrete profile for DNA content analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.
流式细胞术最近已成为一种用于细胞质和核抗原定量分析的有用技术。我们在此报告一种通过流式细胞术同时检测细胞质和核抗原的快速、简单、可重复且灵敏的方法。该技术包括在用2%多聚甲醛固定后，用微波炉加热细胞悬液60秒。经过这种处理后，使用小鼠单克隆抗体（mAb）的标准间接免疫荧光染色，在人骨髓单核细胞系U937（CD68、增殖细胞核抗原和Ki-67）、正常供体和白血病患者的外周血白细胞（CD68、脂皮质蛋白-1和增殖细胞核抗原）以及大鼠系膜细胞系1097（结蛋白、α-平滑肌肌动蛋白）上检测到了多种细胞质和核抗原。与目前常用的方法相比，该技术有几个优点。首先，微波处理是一种快速、简单且可重复的方法，大大减少了时间和成本支出，使该技术可广泛用于许多诊断和研究目的的流式细胞术分析。其次，微波处理对于同时检测细胞质（CD68、脂皮质蛋白-1、结蛋白、α-平滑肌肌动蛋白）和核（增殖细胞核抗原、Ki67）抗原产生最佳结果。第三，微波处理也为DNA含量分析产生离散的图谱。最后，如通过光散射测量，微波处理能在细胞和碎片之间保持清晰的区分。这项研究表明，微波处理是一种强大的技术，特别适用于流式细胞术分析中许多细胞质和核抗原的检测。
