Immune complexes were analyzed on the flow cytometer, Cytomics FCM 500 from Beckman Coulter 22, Avenue des Nations CS 54359, 93420, Villepinte. It is equipped with 2 lasers 488 nm, 40 mW and 638 nm, 25 mW. It has the ability to analyze events on five colors: 525 nm, 575 nm, 620 nm, 675/695 nm, and 755 nm.
The sheath fluid for the cytometer was IsoFlow™ Sheath Fluid from Beckman Coulter, Part Number 8448010, an isotonic fluid at a pH 7.35-7.65, with Sodium Phosphate Dibasic, Sodium Fluoride, Diethylene Glycol Phenyl Ether, and 2-Phenoxyethanol.
A microparticle gate was established in agreement with the published method for characterization of circulating cell-derived microparticles by preliminary standardization experiments using a blend of size-calibrated 0.3, 0.5, 0.9, and 3 μm fluorescent beads, Megamix®, Biocytex, Marseille, France [27 (link), 28 (link)]. To enlarge the scale of fluorescent beads, in some experiments, Trucount beads (Beckton Dickinson) of 10 μm were added.
A gate for DNA-anti-DNA immune complexes for each series of experiments was built around DNA-anti-DNA immune complexes observed in the aliquot of the pool of 15 SLE serum samples incubated with calf thymus DNA. See below for more details and for compensations.
The sheath fluid for the cytometer was IsoFlow™ Sheath Fluid from Beckman Coulter, Part Number 8448010, an isotonic fluid at a pH 7.35-7.65, with Sodium Phosphate Dibasic, Sodium Fluoride, Diethylene Glycol Phenyl Ether, and 2-Phenoxyethanol.
A microparticle gate was established in agreement with the published method for characterization of circulating cell-derived microparticles by preliminary standardization experiments using a blend of size-calibrated 0.3, 0.5, 0.9, and 3 μm fluorescent beads, Megamix®, Biocytex, Marseille, France [27 (link), 28 (link)]. To enlarge the scale of fluorescent beads, in some experiments, Trucount beads (Beckton Dickinson) of 10 μm were added.
A gate for DNA-anti-DNA immune complexes for each series of experiments was built around DNA-anti-DNA immune complexes observed in the aliquot of the pool of 15 SLE serum samples incubated with calf thymus DNA. See below for more details and for compensations.
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