Antibiotic solution

Sulforhodamine (SRB), Trizma-base and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Foetal bovine serum (FBS), 2 mM L-glutamine solution, antibiotic solution (penicillin (100 U/mL) and streptomycin (0.1 mg/mL)) and trypsin EDTA solution were obtained from Lonza (Verviers, Belgium). Cell culture plastic flasks (75 cm²), as well as 96-well culture plastic plates were purchased from Lonza (Verviers, Belgium).
Phosphate-buffered saline (PBS) and 0.4% trypan blue solution were obtained from POCH (Gliwice, Poland).

ReN cells were loaded into 96 well-plates coated with matrigel (1:100) at a density of 10⁴ cells/well supplemented with differentiation media for neuronal cells (NM). The differentiation media consisted of DMEM/F12 media (Life Technologies, Grand Island, NY, USA) supplemented with 2 mg heparin (Stemcell Technologies, Vancouver, BC, Canada), 2% (v/v) B27 (Life Technologies, Grand Island, NY, USA) and 1% (v/v) antibiotic solution (Lonza, Hopkinton, MA, USA). After three weeks of differentiation, the cells were treated with bacteria and microglia-stimulated conditioned media. The experiment involved four treatment groups and two control groups.