流式细胞仪每秒进样细胞数对细胞G0/G1期峰的荧光强度值及变异系数值的影响

在生物学研究中,利用细胞内DNA双螺旋结构碱基对与某些核酸荧光染料有良好的亲和力,荧光染料在光的激发下,产生特定的荧光,运用流式细胞仪检测、分析其荧光强度(Mean值),及其变异系数(CV值),了解细胞内DNA含量,已成为科学分析细胞DNA含量及其所处周期状态的一种手段[1,2].然而,流式细胞仪对操作者技术要求高,同一样本不同实验室检测的结果不尽相同;即使是同一实验室,不同操作人员检测结果亦不一样.其原因既有各实验室评价指标的差异[2-4],也有操作人员个人习惯以及机器本身性能的差异,选定技术参数不同等等.本研究旨在探讨流式细胞仪每秒进样细胞数对检定细胞G0/G1期峰Mean值及CV值的影响,以寻求合适的每秒进样细胞数.

The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism. Human PMNs and PBMCs were isolated from the venous blood of healthy donors by dextran sedimentation and density gradient centrifugation. In the presence of lipopolysaccharide (LPS), PMNs and PBMCs were cocultured at the ratio of 2:1 for 20 h and the concentration of TNF-α in the supernatant was measured by enzyme-linked immunosorbent assay. The binding rate of monocytes with the fluorescein isothiocyanate-labeled LPS (FITC-LPS) and the mean surface fluorescence intensity of monocytes were analyzed by flow cytometry. Results showed that PMNs were capable of inhibiting the TNF-α release from PBMCs (P＜0.05). PMNs suppressed the TNF-α release from PBMCs by 45% on average when PMNs and PBMCs cocultured at the ratio of 2:1. Paraformaldehyde-fixed PMNs still demonstrated the same inhibition (P＜0.05)，which proved that the inhibition was dependent on cell-to-cell contact and suggested that effector molecules responsible for this effect existed on the cell surface of PMNs. In the presence of PMNs, the binding rate of monocytes with the FITC-LPS and the mean surface fluorescence intensity of monocytes were not affected compared with PBMCs alone (P＞0.05). As incubation time was prolonged, the binding of FITC-LPS to monocytes increased (P＜0.05). Thus PMNs did not block the binding of LPS with monocytes. It was concluded that PMNs suppressed the TNF-α release from PBMCs via cell-to-cell interaction. In a cell-contact dependent manner, PMNs might interfere with the signal transduction pathway through which LPS activated PBMCs, thus attenuating the response of PBMCs to LPS and downregulating the TNF-α release.