Cd3 af700

Immune cells were isolated from whole-brain homogenates as recently described (15 (link)). Briefly, brain tissue was processed using the neural tissue dissociation kit (Miltenyi Biotec) in accordance with the manufacturer’s specifications. Afterward, cells were separated using a discontinuous Percoll density gradient (30–37–70% Percoll layers), which results in the enrichment of all immune cell types and removes a lot of myelin, which is auto-fluorescent. Following density gradient centrifugation, immune cells were washed and stained for 30 min at 4 °C using the following antibodies: CD11b APC-Cy7 (BD Biosciences), CD45.2 Pacific blue, CD86 PE-Cy5, and CD206 PE-Cy7 (BioLegend). Respective isotype control antibodies or unstained samples were used to determine positive populations. Myelin debris and dead cells were excluded by FSC/SSC gating, and singlet populations were analyzed.
For flow cytometric analyses of blood samples, EDTA-blood was stained at 4 °C for 15 min with the following antibodies: B220 Pacific Blue, CD3 AF700, CSF-1R BV605 (BioLegend), and CD11b APC-Cy7 (BD Biosciences). For more details see Waltl et al. (15 (link)).
Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).