Automated CSF Leukocyte Analysis by Flow Cytometry

Automated CSF leukocyte counts were performed on ABX micros 60 (Horiba, Montpellier, France). Cells were washed twice with phosphate-buffered saline and immune-stained for 30 min at 4 °C with the following panel of antibodies: anti-CD45-BV786, anti-CD8-BV510, antiCD66b-BV421, anti-CD45-PerCp5.5 (BD Bioscience, San Jose, CA, USA); anti-CD3-FITC, anti-CD19-PECy7, anti-CD11b-PECy5, anti-CD161-PE, anti-CD14-Alexa700, anti-CD11c-APC (Tonbo Biosciences, San Diego, CA, USA). Optimal antibody concentrations were previously defined by titration^{20 (link)}. The cells were washed and suspended in 1% paraformaldehyde. Flow cytometry data was collected on a BD FACS LSR flow cytometer equipped with four lasers. Analyses of leukocyte subsets on CSF and PB samples were conducted by 13 flow cytometry within 30 min from collection. BD FACS Diva software was used for data acquisition and Flow Jo v10 (Becton–Dickinson Bioscience, San Jose, CA, USA) was used for data analysis. The gating strategy is depicted in Supplemental Fig. 2.