A critical appraisal of factors affecting the accuracy of results obtained when using flow cytometry in stem cell investigations: Where do you put your gates?

Abstract

Flow cytometry is used extensively in stem cell investigations but there is wide variation in the methods used for data analysis between laboratories. Data analysis can be challenging in stem cell biology as there is often no clear distinction between positive and negative populations. We have undertaken a critical appraisal of factors that affect the accuracy of results in stem cell applications. We used mouse embryonic stem cells and determined the expression of three common antigens in stem cell investigations, namely CD15, CD184, and c-kit. We have compared different cell preparation methods and gating strategies and also evaluated the use of isotype controls and unstained cells as controls for the identification of positive populations. The use of a ''doublet discriminator'' using a side scatter area signal versus side scatter height signal dot plot to identify single cells for analysis increases the accuracy of results regardless of the method used to dissociate cells. Isotype controls can be helpful in mimicking cellular nonspecific binding of the experimental antibody reaction. Isotype controls behave differently on stem cells at different stages of differentiation. Analysis of a viable single cell population with careful selection of control cell populations increases the accuracy of results. ' 2009 International Society for Advancement of Cytometry

In search for cross‐reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry

“…Indeed, in the study of Braun et al (13), accutase was used, whereas trypsin/EDTA was used in our study. Trypsin is a pancreatic serine protease, whereas accutase exhibits protease and collagenolytic activities (15). Interestingly, it has been reported that trypsin can cause removal and/or functional impairment of certain cell-surface membrane proteins Similar to CD105, the expression of CD73 on human MSC must exceed 95% according to the ISCT (7).…”

“…However, many flow cytometric techniques have been developed for analyzing mature, nonadherent leukocytes, and therefore, some refinements are required when using stem cells (15). For example, the use of gating strategies is important not only to select the population of interest based on the selected markers but also to ensure an accurate analysis of the obtained data by excluding aggregates.…”

“…For example, the use of gating strategies is important not only to select the population of interest based on the selected markers but also to ensure an accurate analysis of the obtained data by excluding aggregates. Furthermore, isotype controls and/or unstained cells are imperative to make a clear distinction between fluorescent positive and negative populations (15).…”

Phenotypes of stem cells from diverse origin

“…Data analysis can be challenging in stem cell biology as there is often no clear distinction between positive and negative populations (27). In addition, expression levels of stem cell markers are also related with the method of cell isolation (28).…”

Simultaneous characterization of progenitor cell compartments in adult human liver

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