Sample 5
Mus musculus
Protocols: Mice were sacrificed by CO2 inhalation and intracardially perfused with approximately 20 ml PBS. The brain was carefully removed from the skull and the spinal cord was flushed out by hydrostatic pressure. Brain and spinal cord were pooled and cut into small pieces using scissors, followed by a tissue digest in 0.4 mg/ml collagenase type IV (from Clostridium histolyticum) in HBSS (containing Ca2+ / Mg2+) supplemented with 10 % FCS for 45-60 min at 37°C while agitating. Digested tissues were passed through an 18 G syringe and filtered through a 70μm cell strainer to obtain a homogeneous cell suspension. After washing, the cell suspension was further enriched using a 30 % Percoll continuous gradient, centrifuged at 4°C for 30 min at 10'800 rpm with low brakes. The myelin in the upper Percoll layer was removed and the remaining cell suspension was washed and transferred into 1.5ml tubes for further analysis. Single cell suspensions were stained with fluorophore labeled monoclonal antibodies for 20- 30 min at 4 °C in an appropriate amount of PBS or FACS buffer (PBS with 2 % FCS and 0.01 % NaN3). Before sorting the cells were washed once with PBS and filtered through a 70 μm cell strainer again. Cell sorting was carried out on a FACSAria III (BD Biosciences) using an 85 μm nozzle, with a purity of >95 %. Mice were treated with 5 x 2.5mg Tamoxifen ip at 6 to 8 weeks age and sacrificed 10 days later. Sorted cells were lysed in RLT plus buffer (Qiagen) supplemented with 1 % β- mercaptoethanol. RNA isolation was performed using the RNeasy Micro Plus Kit (Qiagen) according to the manufacturer's instructions. For next generation sequencing 2 ng RNA from sorted microglia were amplified using the Ovation® Single Cell RNA-seq System kit (Nugen) according to the manufacturer's protocol.