McCarthy D A, Macey M G
School of Biological Sciences, Queen Mary and Westfield College, London, UK.
J Immunol Methods. 1993 Aug 9;163(2):155-60. doi: 10.1016/0022-1759(93)90117-p.
A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.
已开发出一种新方法，可通过流式细胞术对未裂解、未固定的外周血样本中的中性粒细胞和单核细胞上与功能相关的抗原进行定量分析。用丝氨酸酯酶抑制剂苯甲基磺酰氟抗凝的新鲜抽取血液，在4℃下与用单克隆抗体标记的活性核酸染料LDS - 751混合5分钟，稀释后在五参数流式细胞仪中进行分析。基于三种主要白细胞亚群（中性粒细胞、淋巴细胞和单核细胞）的侧向光散射以及它们在FL3通道中与LDS - 751的染色强度（红细胞和血小板染色非常弱），可实时分辨这三种亚群，同时分别在FL1或FL2通道中监测由于结合异硫氰酸荧光素或藻红蛋白标记抗体而产生的荧光强度。该方法避免了因使用固定剂、红细胞裂解剂或也是二价金属离子螯合剂的抗凝剂而可能出现的潜在假象。它应广泛适用于对那些与功能相关的抗原进行定量分析，例如黏附分子和免疫复合物受体，其表面表达在激活后可迅速上调，同时也适用于对那些表达不受快速调节的白细胞表面抗原进行定量分析。
