Immunopanning purification of astrocytes

To examine responses of human and mouse astrocytes to stressors, we purified human and mouse astrocytes from developmentally equivalent stages, to the best of our knowledge. In humans, astrocytogenesis starts during the second trimester and continues through the third trimester^78–80. Human astrocytes reach maturity roughly around one year of age as determined by gene expression^70–72, although their physiological and functional maturation timeline is unclear. In mice, astrocytogenesis starts at the perinatal period (embryonic day 17.5 [E17.5] of a 19-day gestation) and peaks between P0 and P14⁸¹. Mouse astrocytes reach maturity roughly around one month of age as determined by morphology and gene expression⁸². A single-cell RNA-seq study of human and mouse brains found that molecular features of gestational week 16–20 human brains are similar to P0-P5 mouse brains⁸³. Therefore, we purified astrocytes from gestational week 17–20 human brains and P1–3 mouse brains. Within those age ranges, we did not observe age-dependent differences in any of the assays we tested.

We started astrocyte purification experiments using human and mouse brain tissue within similar postmortem intervals. For human samples, we received tissue within 30 min to 1 h postmortem. We then performed a very simple dissection procedure that takes <3 min. For mouse samples, we combined one to two litters of 8–10 mice to get enough cells for each experiment. We combined both male and female mice for all experiments, although sex may not have been equally represented in each litter of mice. Cerebral cortex dissection from all the mice typically took ~45 min before we started the astrocyte purification experiments. We purified human and mouse astrocytes according to a previously published immunopanning protocol^26,28,41. Briefly, we coated three 150 mm-diameter petri dishes first with species-specific secondary antibodies and then with an antibody against CD45 (BD550539, both human and mouse, 10 μl antibody in 12 ml buffer per panning plate), a hybridoma supernatant against the O4 antigen (mouse, 4 ml hybridoma supernatant in 8 ml buffer per panning plate) or an antibody against CD90 (BD550402, human, 20 μl antibody in 12 ml buffer per panning plate), and an antibody against HepaCAM (R&D Systems, MAB4108, 10 μl antibody in 12 ml buffer per panning plate), respectively. We dissected cerebral cortices from human and mouse in PBS and removed meninges. We then dissociated the tissue with 6 units/ml papain at 34.5°C for 45 min. We mechanically triturated the tissue with 5 ml serological pipets in the presence of a trypsin inhibitor solution. We then depleted microglia/macrophages, oligodendrocyte precursor cells, and neurons from the single-cell suspension by incubating the suspension sequentially on the CD45, O4 (for mouse), or CD90 (for human) antibody-coated petri dishes. We incubated the single-cell suspension on the HepaCAM antibody-coated petri dish. After washing away nonadherent cells with PBS, we lifted astrocytes bound to the HepaCAM antibody-coated petri dish using trypsin and plated them on poly-D-lysine-coated plastic coverslips in a serum-free medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, 11960069), Neurobasal (Life Technologies, 21103049), sodium pyruvate (Life Technologies 11360070), glutamine (Life Technologies, 25030081), N-acetyl cysteine (Sigma, A8199), and heparin-binding EGF-like growth factor (Sigma, E4643). For most of the H₂O₂, TNFα, hypoxia, and poly I:C treatment experiments, with exceptions detailed below, astrocytes were plated on 24-well culture plates at 75–100k per well. Human and mouse astrocyte cultures had similar final densities for every type of experiment. For high-density cultures for H₂O₂ treatment, 30k astrocytes were plated in a 50 μl droplet in the middle of pre-dried poly-D-lysine-coated plastic coverslips on 24-well plates. After allowing the cultures to settle for 20 min at 37°C, additional media were added. For Seahorse Respiration Assays, astrocytes were plated at 100–250k/well in Agilent Seahorse 96-well cell culture microplates (cat#101085-004). For TMRE/MTG imaging, astrocytes were plated at 25–50k/well on dark-walled flat-bottom 96-well assay plates (Corning, cat#3603). For poly I:C treatment, astrocytes were plated directly on poly-D-lysine-coated 24-well culture plates (Fisher, cat#08-772-1) without coverslips because poly I:C addition often causes cell to float away from the coverslips. To purify xenografted human astrocytes and host mouse astrocytes from adult host mouse brains, we dissociated whole brains using 20 units/ml papain, depleted microglia/macrophages, oligodendrocytes, and oligodendrocyte precursor cells with anti-CD45 antibody-, GalC hybridoma supernatant-, and O4 hybridoma supernatant-coated plates, respectively. Three consecutive plates with the same antibody were used for depletion of each cell type. We then collected astrocytes with anti-HepaCAM antibody-coated plates. The general procedures we used for the purification of human and mouse astrocytes are based on a previously developed method for purifying rat astrocytes⁴¹, although we used different versions of antibodies for the isolation of cells from different species.