Percp anti cd45 30 f11

Single cell suspensions were generated from murine tissues of C57/BL6 and BALB/c mice. Afterwards, cells were treated with FcR-blocking reagent (Miltenyi Biotec) for 10 min at 4°C after washing with flow cytometry buffer. To exclude dead cells, samples were washed twice with PBS and incubated with BD Horizon™ Fixable Viability Stain 510 viability dye (for 10 min at RT in the dark). Subsequently surface staining to discriminate immune cell populations with PerCP anti-CD45 (30-F11, BD Biosciences), APC-Cy 7 anti-TCRβ (H57-597, BioLegend), Pe-Cy7 anti-CD11b (M1/70, BD Biosciences), and anti-CD19 (6D5, BioLegend), was performed. The data was acquired using a BD FACS LSR II instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc, version 9.9.6 or 10.4.1).

For FACS analysis, the brains of 24 animals were used (n = 6 per group). CD115 and TLR4 staining was realized using the following antibodies: FITC anti-CD11b (M1/70, Bio-Legend), PerCP anti-CD45 (30-F11, BD Biosciences), PE anti-CD115 (T38-320, BD Biosciences), and APC anti-TLR4 (SA15-21, Bio-Legend). Here, CD11b⁺ CD45⁺ cells were gated and afterwards the percentage of TLR4-positive and CD115-positive cells was calculated. DAPI (1:100, Sigma Aldrich) was added to the cell suspension to exclude dead cells.
All samples were measured using FACS and evaluated with FlowJo software (Tree Star).