Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.
Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.
复制标题
通过激光扫描细胞术对人类实体瘤固定细胞悬浮液进行定量多参数免疫荧光测量的可重复性提高的指南。
DOI：
10.1002/cyto.b.20084
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Kociban,Deborah
中科院分区：
文献类型：
--
作者：
Shackney,Stanley;Emlet,DavidR;Pollice,Agnese;Smith,Charles;Brown,Kathryn;Kociban,Deborah
BackgroundLaser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell‐based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell‐based immunofluorescence measurements.Methods and resultsWe describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell‐based analysis. Improvements in the quality of cell‐based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided.ConclusionsAdvances in methodology can improve cell‐based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. © 2005 Wiley‐Liss, Inc.
