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.
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