Neural tissue dissociation kit postnatal neurons

The primary microglial cells were isolated from the male homo-PKD rats at 3 months of age using magnetic beads from Neural Tissue Dissociation Kit-Postnatal Neurons (Miltenyi Biotec, cat#130-094-802, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The isolated retina was immersed in 6 mL of Dulbecco’s phosphate buffered saline (D-PBS) (Sigma, Taufkirchen/Munich, Germany) and then digested with Enzyme mix 1 (buffer with enzyme P) for 15 min at 37 °C and Enzyme mix 2 for 10 min. The retinal tissue was then dissociated by pipetting the whole solution up and down around 10 times. The cell suspension was loaded onto a MACS SmartStrainer (70 µm) (Miltenyi Biotec). After centrifugation at 1200 rpm for 5 min, the cells were collected and labeled with rabbit anti-CD74 antibody (Santa Cruz, cat#SC-20082, 1:100, Heidelberg, Germany) for 1 hour at room temperature. After sorting with anti-rabbit magnetic beads, the CD74⁺ cells were harvested and seeded in fibronectin-coated T25 flasks and cultured in a MACS Neuro medium (Miltenyi Biotec, cat#130-093-570) in a humidified incubator at 37 °C with 5% CO₂.

To isolate cells from brain PN3‐7, mice were sacrificed, brain tissue was collected, and single cell suspensions were generated using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) for OPC, CD11b⁺ cells (microglia), and astrocyte isolation, or Neural Tissue Dissociation Kit ‐Postnatal Neurons (Miltenyi Biotec) for neuron isolation. Cells were isolated by magnetic separation using CD140a (PDGFRα) MicroBead Kit (Miltenyi Biotec), CD11b (Microglia) MicroBeads (Miltenyi Biotec), anti‐ACSA‐2 MicroBead Kit (Miltenyi Biotec), or Neuron Isolation Kit (Miltenyi Biotec). All steps were done according to the manufacturer's protocols.

Primary neurons were derived from the cerebral cortex of p0–p1 mice following standard operational procedure using the neural tissue dissociation kit—postnatal neurons (Cat. 130-094-802, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), as previously described [30 (link)]. In brief, the brain cortices from 6 mice of both sexes were pooled as a single experimental group and subjected to enzymatic and mechanical dissociation, then 150,000 primary neuronal cells were seeded for each well of a poly-L-ornithine-coated 24-well plate, replacing half of the medium volume every 2 or 3 days. At day 10, 37,500 primary microglia cells isolated from the whole brain of adult male or female mice (age 3–6 months) were seeded on a neuron layer [19 (link)]; briefly, the brains from two mice were pooled and subjected to enzymatic and mechanical dissociation and microglia were purified using a magnetic column and anti-CD11b-coated microbeads (Cat. 130-093-634, Miltenyi Biotec). Neuronal and microglial cultures were grown in Neurobasal A medium (Cat. 10888-022, LifeTechnologies, Carlsbad, CA, USA) containing 1% streptomycin–penicillin, 1% GlutaMAX, 2% B-27 Supplement (Cat. 17504-044; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 10 mM HEPES (Cat. H0887, Merk, Darmstadt, Hesse, Germany), in a humidified 5% CO₂-95% air atmosphere at 37 °C.

Primary brain derived microglia from WT and IL-10 KO mice (2–14 days postnatally, female and male) were isolated via magnetic cell sorting. To perform one experiment 5 mice pubs were used per genotype. In brief, postnatal mice were decapitated, brains were isolated and transferred into ice-cold PBS containing 1% BSA (Carl Roth, Karlsruhe Germany). Brains were minced and enzymatically digested by using Neural Tissue Dissociation Kit – Postnatal Neurons (Miltenyi Biotec, Bergisch Gladbach, Germany). After removing myelin using Myelin Removal Beads II (Miltenyi Biotec), CD11b-positive cells were positively selected by magnetic separation using CD11b (Microglia) MicroBeads, human and mouse (Miltenyi Biotec), according to the manufacturer’s instructions. In total 19 experiments were performed. An average yield of 1.5 ± 0.15 × 10⁶ CD11b+ cells/mouse brain from WT and 1.6 ± 0.17 × 10⁶ CD11b+ cells/mouse brain from IL-10 KO mice were isolated.