Cd16 apcalexafluor750

CSF and EDTA blood samples were obtained from all patients and processed as described previously (19 (link)). Briefly, cells from CSF or 100 μl EDTA blood were incubated in VersaLyse buffer (Beckman Coulter; Brea, CA) for 10 min and subsequently washed three times with PBS supplemented with 2% heat-inactivated FCS and 2 mM EDTA. Following incubation with fluorochrome-conjugated antibodies (CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange, all Beckman Coulter), cells were acquired on a Navios flow cytometer (Beckman Coulter). Analysis was conducted with Kaluza V1.2. Lymphocytes, monocytes, and granulocytes were selected based on forwards scatter channel, sideward scatter channel, CD14, and CD45 expression characteristics. Lymphocytes subsets were selected as CD3⁺CD4⁺ (T helper cells), CD3⁺CD8⁺ (cytotoxic T cells), CD3⁺HLA-DR⁺ (activated T cells), CD3⁻CD56⁺ (NK cells), CD3⁻CD19⁺ (B cells), CD3⁻CD19⁺CD138⁺ (plasma cells), whereas monocyte subsets were selected as CD14⁺CD16⁻ (classical monocytes), CD14⁺CD16⁺ (non-classical monocytes) cells.

Multiparameter flow cytometry of immune cells in PB and CSF samples was done as described previously (24 (link), 29 (link)). During lumbar puncture CSF was sampled into polypropylene tubes. All CSF samples were processed in < 20 min. Cells were isolated from CSF by centrifugation (15 min, 290 g, 4°C) and subsequent incubation in VersaLyse buffer (Beckman Coulter, Germany). PB samples were collected in EDTA monovettes and cells were isolated by using VersaLyse buffer. For immunostainings, the following fluorochrome-conjugated antibodies were used: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APC-Alexafluor700, CD16-APC-Alexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all from Beckman-Coulter). Data acquisition was performed with a Navios flow cytometer (Beckman-Coulter). Gating strategy for Leukocytes and Monocytes is described and illustrated in Supplementary Figure 1.

Single-cell suspensions from human peripheral blood mononuclear cells and CSF cells were stained for 30 min at 4 °C with the appropriate combination of indicated fluorescence-labeled monoclonal antibodies in PBS, containing 0.1% sodium azide and 0.1% bovine serum albumin (BSA) following treatment with VersaLyse (Beckman Coulter GmbH, Krefeld, Germany) according to the manufacturer’s instructions. The following monoclonal antibodies were used at 1:200 dilutions: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). Flow cytometric analysis of stained cells was performed following standard protocols. Cells were analyzed on a Navios™ flow cytometer (Beckman Coulter) using Kaluza Analysis Software (V2.1, Beckman Coulter) and presented using Prism 6.0 (Graph Pad).