Page 65 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

Isolation of peripheral blood mononuclear cells and antibody staining
Peripheral blood mononuclear cells (PBMCs) were cryopreserved using the same protocol in all centres within 24 hours of blood collection. PBMCs were extracted using Ficoll-Paque (GE Healthcare, Chicago, USA) gradient reagent. The isolated PBMCs were subsequently frozen in 10% dimethyl sulfoxide (DMSO; Sigma Aldrich, Saint Louis, USA) in fetal calf serum (FCS; Sigma Aldrich). These samples were stored until use in liquid nitrogen vessels in the participating centres and transported to the Leiden University Medical Center for antibody staining, data acquisition and analysis.
Antibodies
Supplementary table 4.1 lists all heavy metal isotope-tagged monoclonal antibodies that were used. Conjugation of the purified antibodies with heavy metal reporters was performed in-house using the MaxPar X8 Antibody Labeling Kit (Fluidigm, San Francisco, USA) according to the manufacturer’s instructions. Optimal labelling concentration was determined by titration for all antibodies.
Antibody Staining and Data Acquisition
PBMCs were thawed according to the standard protocol used at the Department of Immunohematology and Blood Transfusions of the Leiden University Medical Center. After thawing, cells were washed in MaxPar Cell Staining Buffer (CSB; Fluidigm) and incubated with 1 μM Cell-ID intercalator-103Rh in CSB for 15 min at room temperature. Cells were then incubated for 10 min at room temperature with human Fc receptor blocking solution (BioLegend, San Diego, USA) and subsequently stained with the heavy metal isotope- tagged monoclonal antibody mix mentioned in section 2.3 for 45 min at room temperature. After 3 times washing, cells were incubated with 0.125 μM Cell-ID intercalator-Ir (Fluidigm) in MaxPar Fix and Perm buffer (Fluidigm) overnight at 4°C.
The next day, stained cells were washed, resuspended in de-ionized water and acquired on a Helios-upgraded CyTOF2 and Helios mass cytometer 35 (Fluidigm) at an event rate of <500 events/sec in a soluition containing EQ Four Element Calibration Beads (Fluidigm) that were used for normalization for each experiment.
Immune cell composition in peripheral blood
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