ACD-whole blood collected from the retro-orbital plexus was incubated with an excess of ice-cold lysis buffer plus 1 μM prostaglandin E1 (PGE1) to prevent PLT activation and in vitro formation of PLAs as described.29 (link) The blood cells were lysed briefly (1 min), spun at 4°C and stained with a cocktail of antibodies at 1:100 dilution: CD45-Pacific blue (eBioscience), CD115-APC (eBioscience), Gr-1-PerCP cy5.5 (Ly6-C/G; BD Biosciences), CD11b-PE Cy7 (eBioscience) and CD41-FITC (eBioscience). Platelet Ly-6Chi monocyte aggregates, platelet Ly-6Clo monocyte aggregates and platelet neutrophil aggregates were defined as CD45+Gr-1hiCD115+CD41+, CD45+Gr-1loCD115+CD41+ and CD45+Gr-1+CD115−CD41+.
Protocol detail
Assay for Platelet-Leukocyte Aggregates
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Antibodies cocktail
Blood
Blood cells
Buffer
Cd11b
Cold
Cy5 5
Dilution
Fitc
Monocyte
Neutrophil
Plas
Platelet
Prostaglandin e1
Corresponding Organization :
Other organizations : Columbia University
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Variable analysis
independent variables
- Lysis buffer plus 1 μM prostaglandin E1 (PGE1)
- Platelet Ly-6Chi monocyte aggregates
- Platelet Ly-6Clo monocyte aggregates
- Platelet neutrophil aggregates
- Whole blood collected from the retro-orbital plexus
- Brief lysis of blood cells (1 min)
- Staining with a cocktail of antibodies at 1:100 dilution: CD45-Pacific blue, CD115-APC, Gr-1-PerCP cy5.5, CD11b-PE Cy7, and CD41-FITC
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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