Cd11b vio bright fitc

MHC Class II‐VioBlue (Miltenyi Biotec, Cat.# 130‐112‐237, clone: REA813, 1:50), CD206 (mannose receptor)‐Brilliant Violet 711 (BioLegend, Cat.# 141727, clone: C068C2, 1:40), CD11b‐Vio Bright FITC (Miltenyi Biotec, Cat.# 130‐113‐805, clone: REA592, 1:50), CD45‐PerCP‐Vio 700 (Miltenyi Biotec, Cat.# 130‐110‐663/130‐110‐801, clone: REA737, 1:50), F4/80‐PE‐Vio 770 (Miltenyi Biotec, Cat.# 130‐118‐459, clone: REA126, 1:50), Gr1‐APC (Miltenyi Biotec, Cat.# 130‐112‐307, clone: REA810, 1:50), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:1000).

The purity of the astrocyte cultures was confirmed by flow cytometry. First, cells were blocked with FcR blocking reagent (#130-092-575, Miltenyi Biotec, Madrid, Spain,) in 1× phosphate buffer saline (PBS) and 0.5% bovine serum albumin (BSA) and then stained with combinations of fluorophore-conjugated antibodies purchased from Miltenyi Biotec: for astrocytes, ACSA-2-PE (#130-102-365); for microglia, CD11b-Vio Bright FITC (#130-109-368); and for oligodendrocytes, O4-APC (#130-095-895). Data were acquired on a FACSVERSE cytometer and analyzed using FlowJo v10 software (Ashland, OR, USA). Non-viable cells were excluded based on forward and side scatter profiles and 7-Aminodactinomycin (7AAD) staining (#130-111-568).

All data were acquired using a Cytomics FC 500 flow cytometer operating on CXP software equipped with a dual 488 nm/635 nm (blue/red) laser (Beckman Coulter, Brea, CA, USA). Intracellular staining and staining of cell surface antigens were performed simultaneously using the MACS Inside Stain Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, Inside Fix buffer was added to 1 × 10⁵ cells and then resuspended in PBS. The solution was incubated at room temperature and pressure for twenty minutes, after which the cells were washed twice with PBS via centrifugation for five minutes at 300 g. The supernatant was discarded, and the pellet was resuspended in 100 µL of Inside Perm buffer; cells were stained with fluorochrome-conjugated antibodies at appropriate dilutions. Cells were incubated for fifteen minutes at room temperature and then washed with Inside Perm buffer. The supernatant was discarded, the pellet was resuspended in 400 µL of PBS, then flow cytometric analysis was performed. Antibodies against CD11b-VioBright FITC, CD86-PE-Vio770, and CD68-FITC were obtained from Miltenyi Biotec, (Bergisch Gladbach, Germany), whilst antibodies against CD206-PE and CD163-PE were obtained from eBioscience (San Diego, CA, USA).