Wang Lili, DeRose Paul, Gaigalas Adolfas K
National Institute of Standards and Technology, Gaithersburg, MD 20899.
J Res Natl Inst Stand Technol. 2016 Jun 2;121:264-281. doi: 10.6028/jres.121.012. eCollection 2016.
A procedure will be described to assign to each dyed microsphere a number called the Equivalent number of Reference Fluorophores (ERF). The ERF unit gives the number of reference fluorophores in solution which produce the same fluorescence signal as a single dyed microsphere. In the first step, fluorescence measurements were carried out on serial dilutions of a solution of reference fluorophores. The resulting fluorescence intensities and the corresponding concentrations were used to calibrate the response of the fluorometer. The calibration consisted of establishing a linear relation between the intensities and concentrations. In the second step, the fluorescence intensity from a suspension of microspheres was measured in order to determine the equivalent concentration of reference fluorophores which gave the same fluorescence intensity as the suspension of microspheres. This was performed by utilizing the calibration line obtained in the first step. In the third step, a flow cytometer and a light obscuration apparatus were used to measure the total concentration of microspheres in the suspensions used for the fluorescence measurements. In addition to the total microsphere concentration, the flow cytometer also enabled the measurement of the concentration of a sub population of microspheres which are used to calibrate the fluorescence scale of a flow cytometer. The fourth step utilized the data collected in steps one, two, and three to assign a value of ERF to individual microspheres. The set of microspheres with assigned ERF values will be used to establish a linear fluorescence scale in each channel of a flow cytometer. The discussion will emphasize the estimate of uncertainties in each step of the assignment process.
将描述一种程序，为每个染色微球赋予一个称为参考荧光团等效数（ERF）的数值。ERF单位给出溶液中产生与单个染色微球相同荧光信号的参考荧光团数量。第一步，对参考荧光团溶液的系列稀释液进行荧光测量。所得荧光强度和相应浓度用于校准荧光计的响应。校准包括建立强度与浓度之间的线性关系。第二步，测量微球悬浮液的荧光强度，以确定产生与微球悬浮液相同荧光强度的参考荧光团等效浓度。这通过利用第一步获得的校准曲线来完成。第三步，使用流式细胞仪和光阻仪测量用于荧光测量的悬浮液中微球的总浓度。除了微球总浓度外，流式细胞仪还能够测量用于校准流式细胞仪荧光刻度的微球亚群的浓度。第四步利用在第一步、第二步和第三步中收集的数据为单个微球赋予ERF值。具有指定ERF值的微球集将用于在流式细胞仪的每个通道中建立线性荧光刻度。讨论将强调赋值过程各步骤中不确定度的估计。
