Gating strategy for both CD14+ monocyte purification and pan-monocyte purification. Cell viability was checked by using PI staining. Monocytes were stained with CD3, CD14, CD19, CD56 and CD66b antibody for both before and after purification samples. Figure S2. Identification of peripheral blood monocyte subsets by flow cytometry. Monocyte subsets were identified by negative selection. Neutrophils, NK cells, B and T cells were excluded by using conventional bivariate scatterplots of side scatter signal versus cell-specific markers. The remaining population was selected with HLA-DR, and was then sub-classified into three monocyte subsets using CD14 versus CD16. Graphs were created using Flowjo software. Figure S3. Percentage of IL-6 positive cells in non-classical monocytes from healthy controls (HC), MS, and NMOSD patients (n = 15). The percentage of IL-6 positive cells in the non-classical monocyte population was calculated for HC, MS, and NMOSD. Graphs were created using Flowjo software. Assessment of statistical significance was performed by two-way ANOVA followed by Dunnettâs multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
