Single-cell suspension

Hippocampi were rinsed in ice-cold D-PBS containing calcium, magnesium, glucose, and pyruvate (#14,287–072, Thermo Fisher Scientific), sliced into four sagittal sections on a chilled, metal block and placed into ice-cold gentleMACS C-tubes (#130–093-237, Miltenyi Biotec), containing 1950 μl of papain-based Enzyme Mix 1. For each reaction, Enzyme Mix 1 was created by combining 50 μl of Enzyme P with 1900 μl of buffer Z, while Enzyme Mix 2 was created by combining 10 μl of Enzyme A with 20 μl of buffer Y per reaction, with all reagents included in the adult brain dissociation kit (#130–107-677, Miltenyi Biotec). Transcription and translation inhibitors were included during cell preparation to prevent ex vivo activational artifacts, as previously described[66]. Actinomycin D (#A1410, MilliporeSigma) was reconstituted in DMSO at 5 mg/ml before being aliquoted and stored at – 20 °C protected from light. Triptolide (#T3652, MilliporeSigma) was reconstituted in DMSO to a concentration of 10 mM before being aliquoted and stored at − 20 °C protected from light. Anisomycin (#A9789, MilliporeSigma) was reconstituted in DMSO to a concentration of 10 mg/ml before being aliquoted and stored at 4 °C protected from light. Two microliter each of actinomycin D, triptolide, and anisomycin stocks was added to the initial Enzyme Mix 1 before dissociation for a final concentration of 5 μg/ml, 10 μm, and 10 μg/ml, respectively. Each sample had 30 μl of Enzyme Mix 2 added before being mechanically dissociated for 30 min at 37 °C on the gentleMACS Octo dissociator with heaters (#130–096-427, Miltenyi Biotec) using the 37C_ABDK_02 program. Following enzymatic and mechanical dissociation, the C-tubes were quickly spun in a chilled (4 °C) Allegra-30R centrifuge (#{"type":"entrez-nucleotide","attrs":{"text":"B08708","term_id":"2089828","term_text":"B08708"}}B08708, Beckman Coulter) with an SX4400 swinging bucket rotor to collect the samples in the bottom of the tubes. Next, samples were resuspended, passed through a pre-wet 70 -μm MACS SmartStrainer (#130–110-916, Miltenyi Biotec), and collected in a 50-ml conical tube (#21,008–178, VWR International). The C-tubes were washed with 10 ml of ice-cold D-PBS, and the washed volume was passed through the 70 -μm MACS SmartStrainer. The cells were then pelleted by centrifugation at 300 × g for 10 min at 4 °C. Following centrifugation, the supernatant was aspirated and debris was removed using the debris removal solution (#130–109-398, Miltenyi Biotec) provided in the adult brain dissociation kit (#130–107-677, Miltenyi Biotec). Briefly, cells were resuspended in 1.55 ml of ice-cold D-PBS, and passed to a 5-ml round bottom tube (#22,171,606, FisherScientific), and 450 μl of cold debris removal solution was mixed into the cell suspensions. Next, 2 ml of D-PBS was gently overlaid on the cell suspension, ensuring the layers did not mix. Centrifugation at 3000 × g for 10 min at 4 °C separated the suspension into three phases, of which the top two phases were aspirated. The cell pellet was gently resuspended in 5 ml of ice-cold D-PBS before centrifugation at 1000 × g for 10 min at 4 °C. After aspirating the supernatant completely, the cells were resuspended in 1 ml 0.5% BSA (#130–091-376, Miltenyi Biotec) in D-PBS and filtered through a 35-μm filter (#352,235, Fisher Scientific). A 100 μl aliquot of cells was retained as “cell-input” for comparison [66].