Neural tissue dissociation kit p

The reagents used in this study were purchased as described: THC and CBD from Cayman Chemical (Michigan, USA), myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅) peptide H-MEVGWYRSPFSRVVHLYRNGK-OH (PolyPeptide Laboratories, San Diego, CA, USA). Mycobacterium tuberculosis (strain H37Ra) (BD, Franklin Lakes, NJ, USA), complete Freund's adjuvant (Fisher, Hampton, NH, USA), Pertussis toxin (List Biological Laboratories, Campbell, CA, USA), Percoll, GE Healthcare Life Sciences (Pittsburgh, PA, USA); Neural Tissue Dissociation Kit (P) (Miltenyi Biotech, Auburn, CA, USA), RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640, l-glutamine, HEPES, phosphate-buffered saline, and fetal bovine serum (VWR, West Chester, PA, USA), ELISA Max Kits IL-10, IL-17A, IFN-γ, IL-6, IL-1β, TNF-α, and TGF-β and FITC Annexin V/-PI apoptosis kit (Biolegend, San Diego, CA). EasySep PE selection kit (Stemcell Technologies, Cambridge, MA, USA), Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA), miRNeasy Mini Kit, miScript II RT Kit and miRNAs primers (Qiagen, Valencia, CA), mRNAs primers (Integrated DNA technologies, Coralville, IA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA).

The mouse retinas were incubated at 37 °C for 5 min and maintained in a humidified atmosphere of 5% CO₂ with a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cell digestion was performed with Gentl MACS according to the manufacturer’s protocol. Incubation time was as follows: m_brain_01: 3 min, m_brain_02: 5 min, and m_brain_03: 5 min; this was followed by additional incubation for 5 min. Single cells were prepared with a cell strainer (40 µm). Manipulation of the primary retinal cultures was performed as previously described, with minor modifications^{44 (link), 62 (link)}. Cell density was adjusted to 1.5 × 10⁵ cells/50 µl in each well of a 96-well plate (Falcon, NY, USA), and the cultures were incubated for 15 min in a CO₂ incubator at 37 °C. The culture medium was neurobasal A (Invitrogen, Carlsbad, CA, USA), containing a B27 supplement without anti-oxidants (NBA/B27AO-, Invitrogen), as well as 1 µg/ml insulin, 2 mM L-glutamate and 12 µg/ml gentamicin. Fifteen minutes after the retinal cells were harvested, hesperidin was applied to the primary retinal cells. Two hours later, Alamar blue (Invitrogen) was added. Eighteen hours later, the fluorescence intensity was measured (560-nm excitation and 590-nm emission) with an absorption spectrometer (Vmax; Molecular Devices, Sunnyvale, CA, USA).

Brains from P7 mouse pups were dissociated into a single-cell suspension using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) according to the manufacturer’s protocol. Microglia were isolated from the single-cell suspension using CD11b microbeads (Miltenyi Biotec) and cultured in DMEM/F12 with 10% FBS (73 (link)). Cells were stimulated with LPS (100 ng/mL) for 18 hours and then washed and incubated with fresh medium containing TGF-β or ACMs.

Brain cells were obtained from new born mice (<2-week-old) using a Neural Tissue Dissociation Kit (P) (Milteny Biotec, Cologne, Germany), treated with CD11b antibody-conjugated microbeads (Milteny Biotec), and then isolated by magnetic cell sorting as CD11b⁺ cells, as previously described^{6 (link)}. The isolated cells were plated on poly-D-Lysine (PDL)-coated 96-well plates (BD Biosciences, CA, USA), and then cultured in DMEM/F12 (Gibco, CA, USA) with 10% fetal bovine serum (FBS; Gibco) containing 100 U/ml of penicillin/streptomycin (Sigma-Aldrich, MO, USA).

Immediately after CSF sampling, brains were harvested and microglia were isolated using the Magnetic-Activated Cell Sorting (MACS) technique, following the manufactures instructions (Neural Tissue Dissociation Kit (P), MACS Miltenyi Biotec), with slight modifications similarly to a previously published protocol [19 ]. In brief, cerebellum, olfactory bulb, and meninges were removed, and each hemisphere was cut into pieces. Dissected brains were homogenized using an automatic (gentleMACS Dissociator) and enzymatic (Enzyme A (10 µl) + Enzyme P (50 µl)) dissociation process. The homogenates were rinsed through a cell strainer (40 μm, Falcon). Filtered samples were pelleted, washed and resuspended in Hanks’ buffered salt solution (HBSS) supplemented with 7mM HEPES. After washing, cells were magnetically labeled with CD11b (Microglia) Microbeads (Miltenyi Biotec) and diluted in 90 µl MACS buffer (PBS supplemented with 5% BSA). The magnetically labeled cell suspension was loaded onto a MACS LS-column, washed with MACS buffer, and eluted into a Protein LoBind tube. The eluted microglia fraction was pelleted and washed with HBSS to remove any remaining BSA, before all liquid was aspirated and the remaining cell pellet was snap-frozen and stored at -80 °C.