Anti cd11b antibody

Microglial cells were isolated from the lumbar spinal cord of mice using CD11b microbeads (Miltenyil Biotec, Germany). Briefly, spinal cords were dissected after perfusion with PBS and single-cell suspensions were prepared by using the neural tissue dissociation Kit (Miltenyil Biotec) for 30 min at 37 °C. Pure single-cells were acquired by passing the cells through a 40 μm strainer. Myelins were removed by using myelin removal beads (Miltenyil Biotec) for 15 min at 4 °C. Single cells were stained with anti-CD11b antibody (Miltenyi Biotec) for 30 min at 4 °C. The CD11b⁺ labeled single cells were separated by a magnetic field through MS columns (Miltenyi Biotec). We confirmed that these cells were approximately 95 % pure, as assessed by CD11b⁺ and CD45⁺ cytometry analysis. Isolated microglia cells were used in real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis to investigate the level of IKKβ deletion in spinal microglia, as previously described [26 (link)], using the primer summarized in Additional file 1.

CD8+ cells in DLN were isolated by magnetic beads conjugated with an anti-CD8 antibody (Miltenyi Biotec) as described in our previous report [36 (link)]. BALF cells were sorted by CD11b+ status using magnetic beads conjugated with an anti-CD11b antibody (Miltenyi Biotec). The magnetically labeled cells were purified using quadroMACS system (Miltenyi Biotec).

Mixed glial cell cultures were prepared from newborn C57BL/6J mouse brains (Harlan, The Netherlands) as previously described [34 (link)]. Cells were plated and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was changed after 3 days of culture. After 12-14 days, mixed glial cell cultures had reached confluence. Microglia present in the astrocyte monolayer were collected by a positive selection using a magnetic cell sorting (MACS) approach, as previously described [34 (link)]. Cells were selected using an anti-CD11b antibody following the manufacturer’s instructions (Miltenyi Biotec, The Netherlands). Finally, microglia were plated in a mix (1:1 v/v) of DMEM and mixed glial cell culture-conditioned medium at 37°C in a humidified atmosphere containing 5% CO₂. Microglial cultures were treated 24 h later. Cell culture products were provided by Invitrogen (Belgium).