Abstract
High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.
Highlights
High-resolution flow cytometers have become available, enabling the detection of extracellular vesicles (EV) in various biological fluids
By using the EV gates established in correspondingly tested five different specific their matched isotype controls combined in two different panels (P1 or panel 2 (P2))
We have focused on creating a more informed basis for deciding how to pre-treat labels in order to minimize positive event counts due to fluorescent label aggregates in samples
Summary
High-resolution flow cytometers (hFCM) have become available, enabling the detection of extracellular vesicles (EV) in various biological fluids. EVs are small (50–1000 nm) membrane-encapsulated particles that are released from cells by various stimuli specific to cell type and disease pathophysiology [1]. EVs are representative of their parent cell and express membrane proteins that are specific to the parent cell and represent the biological state of that particular cell [1]. Flow cytometry is a widely used method for the characterization of EV populations [2]. Conventional flow cytometry lacks the sensitivity to capture EV populations of small sizes (
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