Details
| Original language | English |
|---|---|
| Article number | 6419 |
| Journal | Scientific reports |
| Volume | 14 |
| Issue number | 1 |
| Publication status | Published - 17 Mar 2024 |
| Externally published | Yes |
Abstract
Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.
Keywords
- Extracellular vesicles, Flow cytometry, Fluorochrome-to-protein ratio, Tissue factor
ASJC Scopus subject areas
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In: Scientific reports, Vol. 14, No. 1, 6419, 17.03.2024.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles
AU - Weiss, René
AU - Mostageer, Marwa
AU - Eichhorn, Tanja
AU - Huber, Silke
AU - Egger, Dominik
AU - Spittler, Andreas
AU - Tripisciano, Carla
AU - Kasper, Cornelia
AU - Weber, Viktoria
N1 - Publisher Copyright: © The Author(s) 2024.
PY - 2024/3/17
Y1 - 2024/3/17
N2 - Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.
AB - Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.
KW - Extracellular vesicles
KW - Flow cytometry
KW - Fluorochrome-to-protein ratio
KW - Tissue factor
UR - http://www.scopus.com/inward/record.url?scp=85187918732&partnerID=8YFLogxK
U2 - 10.1038/s41598-024-56841-5
DO - 10.1038/s41598-024-56841-5
M3 - Article
C2 - 38494537
AN - SCOPUS:85187918732
VL - 14
JO - Scientific reports
JF - Scientific reports
SN - 2045-2322
IS - 1
M1 - 6419
ER -