F4 80 apc cy7 clone bm8

On day 28 post tumor cell inoculation, mice were terminated and the brains of the treatment groups and one tumor-free (normal) littermate were dissected and immediately transferred in ice-cold HBSS. Brains were digested in a buffer solution containing HBSS, Collagenase P (0.2 mg/mL), Dispase II (0.8 mg/mL), DNase I (0.01 mg/mL), and Collagenase A (0.3 mg/mL) for 60 min at 37°C under gentle rocking as described before.⁴¹ (link) Briefly, the digested cells were pre-incubated with rat anti-mouse FccIII/II receptor (CD16/CD32) blocking antibody for 5 min at 4°C, and then stained with the fluorochrome-conjugated antibodies (1:100). PI (1:3,000; Thermo Fisher Scientific) was added for live gating. Flow cytometry was performed using FACSDiva software on a LSRII (BD Biosciences; San Jose, CA, USA) and analyzed using FlowJo software (Tree Star; Ashland, OR, USA).
The cells were stained with anti-mouse antibody cocktail: CD45 AF700 (clone 30-F11, eBioscience), Gr-1 PerCP Cy5.5 (clone RB6-8C5, BioLegend), CD11b V500 (clone M1/70, BD), CD11c PE Cy7 (clone N418, BioLegend), F4/80 APC Cy7 (clone BM8, BioLegend), CD206 BV421 (clone C068C2, BioLegend), MHC class II PE-Cy5 (clone M5/114.15.2, eBioscience), T cell receptor (TCR)-β FITC (clone H57-597, BioLegend), and NK1.1 APC (clone PK136, BioLegend).