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目的建立使用流式细胞仪检测血小板跨膜电位的方法,观察血小板在体外保存期间跨膜电位变化。方法使用电压敏感染料Di BAC4(3)对20(人)份血小板(20 m L/份)染色后,用流式细胞仪测定血小板在静息状态下和完全去极化时的荧光强度,根据能斯特方程计算血小板跨膜电位,并利用此方法观察血小板在体外保存期间跨膜电位的变化情况。结果 20人份新鲜血小板跨膜电位平均值为(-56±4)m V,CV=5.5%,与膜电位钳法得到的结果(-50-60)m V相似,保存1、3、5 d的血小板跨膜电位分别为(-56±4)、(-54±8)和(-46±6)m V,其中血小板跨膜电位在血小板保存1 d时明显高于5 d(P<0.05)。结论所建立起的方法用于测定血小板跨膜电位准确、可靠。
Objective To establish a method for the detection of platelet transmembrane potential by flow cytometry and observe the change of transmembrane potential during the in vitro preservation of platelets. Methods Twenty (human) platelets (20 m L / part) were stained with the voltage-sensitive dye Di BAC4 (3) and the fluorescence intensity of platelets at resting and complete depolarization was measured by flow cytometry. Nernst equation was used to calculate platelet transmembrane potential, and the use of this method to observe platelet transmembrane potential changes during in vitro preservation. Results The mean transmembrane potential of 20 fresh platelets was (-56 ± 4) mV, CV = 5.5%, which was similar to that obtained with membrane potential-clamp method (-50-60) (-56 ± 4), (-54 ± 8) and (-46 ± 6) mV, respectively. The platelet transmembrane potential was significantly higher than that on the 1st day of platelet preservation (P < 0.05). Conclusion The established method is used to determine the platelet transmembrane potential is accurate and reliable.