Hla dr ecd clone immu 357

Flow cytometric measurements were done in one study centre (Muenster). For this purpose, PB and CSF were analysed within one hour after sampling. The CSF was centrifuged and treated with VersaLyse^TM (Beckman Coulter, Krefeld, Germany) in parallel to PB, according to the manufacturer’s instructions. Cells were incubated with fluorochrome-conjugated monoclonal antibodies [CD14-FITC (clone RM052), CD138-PE (clone B-A38), HLA-DR-ECD (clone Immu-357), CD3-PC5.5 (clone UCHT1), CD56-PC7 (clone N901), CD4-APC (clone 13B8.2), CD19-APC A700 (clone J3-119), CD16-APC A750 (clone 3G8), CD8-PacificBlue (clone B9.11), CD45-KromeOrange (clone J.33); all Beckman Coulter, dilution 1:200] for 30 min, washed and analysed by flow cytometry on a Navios^TM (Beckman Coulter) flow cytometer.³² (link) Data were analysed with Kaluza^TM 2.1 software (Beckman Coulter; for gating strategy, see Supplementary Fig. 1). Routine CSF parameters were investigated in addition to flow cytometry in both study centres (Magdeburg and Muenster). Cells were counted using a Fuchs-Rosenthal chamber. The CSF/serum IgG, IgA, IgM, and albumin ratio and the blood/CSF-barrier integrity were determined by nephelometry (BN ProSpec^TM, Siemens Healthcare). IgG oligoclonal band (OCB) patterns were analysed by isoelectric focussing in gel-electrophoresis and subsequent silver staining (Processor Plus^TM, GE Healthcare).

Flow cytometry analysis was performed on the maximum of recovered cells from the AqH samples (≤10⁶ cells). Cells were first blocked with FcR anti-human blocking reagent (Miltenyi). Afterwards, cells were stained for 30 min at 4°C in the dark with a combination of the following anti-human antibodies: CD3 (perCp-Cy5.5, clone OKT3), CD4 (BV510, clone OKT4), CD8 (APC, clone SK1), CD11b (FITC clone M1/70), CD11c (Pacific Blue, clone 3.9)—all from Biolegend—and CD56 (Pe-Cy7, clone N901) and HLA-DR (ECD, clone Immu-357) from Beckman Coulter. Samples were measured on a Gallios (10 colors, 3 lasers; Beckman Coulter) flow cytometer using FACS Kaluza software v2.1.1 (Beckman Coulter). Data were analyzed with FlowJo v10.6.1 (BD Biosciences). The gating strategy is illustrated in Figure 2—figure supplement 2.

Whole blood was stained with LIVE/DEAD fixable violet stain (ThermoFisher Scientific, Montigny le Bretonneux, France), CD3-PerCP (clone SP34-2, BD Biosciences, Le Pont-de-Claix, France), CD20-PE (clone 2H7, BD Biosciences, Le Pont-de-Claix, France), HLA-DR-ECD (clone immu357, Beckman Coulter, Villepinte, France), CD8-PECy7 (clone RPA-T8, BD Biosciences, France), CD14-APC (clone TÜK4, Miltenyi Biotec, Paris, France) and CD16-APC-AF750 (clone 3G8, Beckman Coulter, Villepinte, France). Monocytes and PMNs were analyzed in whole blood after fixation with paraformaldehyde (1%). Cells were analyzed using an Attune NxT acoustic focusing cytometer (ThermoFischer Scientific, Montigny le Bretonneux, France) with a violet scatter filter (NxT No-Wash No-Lyse filter) installed with a collection rate of 200 µL/min. Analyses of two time points (4 and 24 h) were performed at the same time in order to attain comparative analyses of fluorescence intensities.