Flow cytometry of MVs and cells was performed according to our published methods (Liu et al., 2007 (link); Li et al., 2010 (link), 2013 (link)). In brief, culture supernatants were fixed in filtered 1% paraformaldehyde (PFA), and supplemented with counting beads. The number of MVs in each sample was quantified using a FACSCalibur flow cytometer, with gating criteria based on particle size and surface exposure of PS, detected by PE-labeled annexin-V (BD Pharmingen) staining (Liu et al., 2007 (link); Li et al., 2010 (link), 2013 (link)). The portion of PS-positive MVs that were also HMGB1-positive was quantified by simultaneous staining with mouse anti-human HMGB1 antibody (R&D), followed by FITC-labeled goat anti-mouse IgG secondary antibody (Abcam). After treatment without or with TSE, adherent monolayers of macrophages were detached and fixed by suspension in 1% PFA for detection of cell-surface HMGB1 with flow cytometry (Li et al., 2013 (link)).
Protocol detail
Flow Cytometry Analysis of Microvesicles
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After treatment
Annexin v
Anti antibody
Anti igg
Antibody
Cell
Fitc
Flow cytometry
Goat
Hmgb1
Human
Macrophages
Membrane
Microvesicles
Mouse
Paraformaldehyde
Corresponding Organization : Philadelphia VA Medical Center
Other organizations : Tianjin Medical University, Second Hospital of Tianjin Medical University, University of Gothenburg
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Variable analysis
independent variables
- Treatment with TSE
- Number of PS-positive MVs
- Portion of PS-positive MVs that are HMGB1-positive
- Cell-surface HMGB1 on macrophages
- Particle size and surface exposure of PS, detected by PE-labeled annexin-V staining
- Simultaneous staining with mouse anti-human HMGB1 antibody and FITC-labeled goat anti-mouse IgG secondary antibody
- Positive control: Adherent monolayers of macrophages, with and without TSE treatment
- Negative control: Not explicitly mentioned
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