Miltenyi gentle macs cell dissociator

Brains were harvested from WNV NY99 and WNV Eg101 infected mice on day 8 after inoculation and homogenized using a Miltenyi gentle MACS cell dissociator (Miltenyi Biotec) and enzymatic neural dissociation kit (Miltenyi Biotec). Infiltrated leukocytes were isolated by discontinuous Percoll gradient centrifugation. Cells were counted and washed once with PBS. Cells were stained for CD45, CD3, NK1.1, CD11b, and Ly6c by directly conjugated antibodies (eBiosciences) for 30 min at 4 °C and then fixed with 2 % PFA. Samples were analyzed by multi-color flow cytometry using FACSAria, and data were analyzed using FlowJo software [19 , 23 ]. Briefly, after gating live cells by FSC and SSC parameters, CD45^hi leukocytes were selected. CD45^hi leukocytes were further divided into CD3⁺, CD11b⁺, and NK1.1⁺ cells as described above [23 ].

The brains and spleens from three WT and db/db mice (two independent experiments, a total of six mice per group) were isolated, pooled and homogenized using a Miltenyi gentle MACS cell dissociator (Miltenyi Biotec, San Diego, California, United States). Infiltrated leukocytes were isolated by discontinuous Percoll gradient centrifugation and quantitated from the brain of WT and db/db mice as described previously [34 (link),37 (link)]. Cells were counted and washed with 1 × fluorescence activated cell sorter buffer (0.5% BSA and 2 mM ethylenediaminetetraacetic acid). To identify for CD45⁺, CD11b⁺, CD3⁺, CD4⁺ and CD8⁺ cell populations, cells were stained using fluorescein isothiocyanate (FITC)-conjugated anti-CD45, phycoerythrin (PE) Cy7-conjugated anti-CD11b, PE-conjugated anti-CD3, PE Texas Red-conjugated anti-CD4 and allophycocyanin (APC)-conjugated anti-CD8 antibodies (eBiosciences, San Diego, California, United States) for 30 minutes at 4°C and then fixed with 4% PFA at 4°C for 15 minutes. Fluorescence minus one samples were prepared for detecting any spillover from other channel. Samples were analyzed by multi-color flow cytometry using FACS Aria and data were analyzed using FlowJo software (version 9.4.11) (TreeStar, Ashland, Oregon, United States) as described previously [34 (link),37 (link)].