TY - JOUR
T1 - Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry
AU - Hajji, Najat
AU - Hau, Chi M.
AU - Nieuwland, Rienk
AU - van der Pol, Edwin
N1 - Funding Information: We would like to acknowledge Dr. Aleksandra G?secka for commenting on the sections about blood collection, plasma preparation, and plasma storage and Naomi Buntsma, M.Sc., for the data on CD45-APC titrations. E. van der Pol acknowledges funding from the Netherlands Organisation for Scientific Research-Domain Applied and Engineering Sciences (NWO-TTW), research programmes VENI 15924, and from the European Association of National Metrology Institutes, Grant/Award Number: 18HLT01 METVES II. Funding Information: We would like to acknowledge Dr. Aleksandra Gąsecka for commenting on the sections about blood collection, plasma preparation, and plasma storage and Naomi Buntsma, M.Sc., for the data on CD45-APC titrations. E. van der Pol acknowledges funding from the Netherlands Organisation for Scientific Research - Domain Applied and Engineering Sciences (NWO-TTW), research programmes VENI 15924, and from the European Association of National Metrology Institutes, Grant/Award Number: 18HLT01 METVES II. Publisher Copyright: © 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022
Y1 - 2022
N2 - Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.
AB - Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.
KW - Calibration
KW - Extracellular vesicles
KW - Flow cytometry
KW - Fluorescent antibody labeling
KW - Human blood plasma
KW - Number concentration
KW - Standardization
UR - http://www.scopus.com/inward/record.url?scp=85128801603&partnerID=8YFLogxK
U2 - https://doi.org/10.1007/978-1-0716-2341-1_5
DO - https://doi.org/10.1007/978-1-0716-2341-1_5
M3 - Article
C2 - 35467279
SN - 1064-3745
VL - 2504
SP - 55
EP - 75
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -