Astrocytes were purified by immunopanning from P5 rat (Sprague Dawley rats obtained from Charles River) forebrains of both sexes and cultured as previously described (). Briefly, cortices were enzymatically (papain) then mechanically dissociated to generate a single cell suspension that was incubated on successive negative immunopanning plates to remove microglia, endothelial cells, and oligodendrocyte lineage cells before positively selecting for astrocytes with an Itgb5-coated panning plate. Isolated astrocytes were cultured in a defined, serum-free base medium containing 50% neurobasal, 50% DMEM, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 292 μg/mL L-glutamine, 1X SATO and 5 μg/mL of N-acetyl cysteine. This medium was supplemented with the astrocyte-required survival factor HBEGF (Peprotech, 100-47) at 5ng/mL as previously described (). Microglia were purified by dounce homogenization on ice and grown in serum-free base medium containing DMEM/F12 containing 100 units/mL penicillin, 100 mg/mL streptomycin, 2mM glutamine, 5 mg/ml N-acetyl cysteine, 5 mg/mL insulin, 100 mg/mL apo-transferrin, and 100 ng/mL sodium selenite, all from GIBCO or Sigma. This medium was supplemented with the microglia-required survival factors: human TGFβ2 (2 ng/mL, Peprotech), murine IL34 (100 ng/mL, R&D Systems), and ovine wool cholesterol (1.5 mg/mL, Avanti Polar Lipids) (). Both astrocyte and microglia cultures were treated with methotrexate dissolved in DMSO for 24 hr, before collection and microfluidic analysis of activation (see below).

Antibody stains were washed out and cells were resuspended in cold HBSS with 1:1000 Near IR LIVE/DEAD (Life Technologies, L10119). Following a 20 to 30 min incubation on ice to label dead cells, samples were washed once with cold HBSS, resuspended in FACS Buffer, and sorted on a BD FACS Aria by sequential yield and purity sorts. OPCs were isolated as the CD140a + (PDGFRα + ) population, microglia as the CD11b + population, and astrocytes as the eGFP + population. Microglia and astrocytes were sorted directly in RLT Plus Lysis Buffer.

For OPC transplantation sorts or OPC IC, whole forebrains were removed from P6-8 GFP(C57BL/6-Tg(CAG-EGFP)1Osb/J; The Jackson Laboratory) or CD57BL/6:CD1 mice pups, respectively, of both sexes. For microglia and astrocyte sorts, frontal deep cortex and subjacent corpus callosum was microdissected from P63-65 mice. Tissue was then minced in Hibernate-A to approximately 1 mmpieces and pelleted at 200xg for 2 min. Supernatant was removed and tissue was resuspended in 25 μg/mL Liberase-H (Roche, 05401054001) solution in HBSS with Ca/Mg. Tissue was enzymatically dissociated for 30 min, rotating at 37°C. Samples were then triturated, and passed through a 100 μm filter. Dissociated tissue was pelleted then resuspended in 30% sucrose/HBSS solution and centrifuged at 2000 rpm for 10 min at 4°C without brake to remove myelin debris. Pellets were washed once in cold HBSS without Ca/Mgand resuspended in Neurobasal-A + Hoechst 33342 (10 μg/ml final). Cells were then incubated at 37°C for 30 min to label nuclei. Samples were pelleted and resuspended in FACS Buffer (2% BSA/HBSS + 10 mM HEPES). Samples were blocked using rat IgG (8 μg/mL final, R&D Systems, 6-001-A) for 10 min on ice and then stained with preconjugated antibodies for 30 to 45 min. For OPC sorts, cells were stained with anti-CD140a (APA5, BioLegend, 135902). For microglial sorts, anti-CD11b (M1/70, BioLegend, 101216) was used. For astrocyte sorts, ALDH1L1-eGFP reporter mice were used (Tg(Aldh1l1-eGFP)OFC789Gsat/Mmucd mice were backcrossed onto a C57BL/6 black background () a gift from the B. Barres and S. Liddelow).

Neural precursor cells (NPCs) were cultured as followed: Base cell medium for neural precursor cells was made using Neurobasal(-A) (Invitrogen, 10888-022), antibiotic-antimycotic (Invitrogen, 15240096), and B27(-A) (Invitrogen, 12587010). Mouse neural precursor cells (NPCs) of low passage number (10-15) were acquired. These cells were thawed from −80°C in a 37°C water bath and transferred to a 15 mL centrifuge tube where 10 mL of Hank’s Balanced Salt Solution without Calcium and Magnesium (HBSS, Fischer Scientific) was added to dilute the cryogenic protecting agent, DMSO. Cells were centrifuged for 5 min (300 g). The supernatant was removed and replaced with 10 mL of base cell medium. This cell mixture was transferred to a T75cm 2 flask (Nunc™ Cell Culture Treated 75 cm 2 EasYFlask™, Fisher Scientific) along with Epidermal Growth Factor (at 20 ng/mL complexed to Alexa Fluor 647 Stretavidin, Life Technologies, E-35351), Fibroblast Growth Factor (20 ng/mL Stretavidin, PeproTech, 450-33), and heparin (2 ng/mL, Stem Cell Technologies, 07980). The NPCs were then used for IC 50 experiments. Mouse OPCs were obtained by FACS (see below) and cultured in DMEM with Glutamax (Invitrogen, 35050-061), sodium pyruvate (Thermo Fisher, 11360070), MEM NEAAs (Invitrogen, 11140050), antibiotic-antimycotic with N21-MAX (R&D systems, AR012) and Trace Elements B (Corning, 25-022-CI) (all the aforementioned supplemented to 1X) and Insulin (5 μg/mL, Sigma, 19278), N-acetyl cysteine (5 μg/mL, Sigma, A9165), PDGFAA (10 ng/mL, PeproTech, 315-17), CNTF (10 ng/mL, PeproTech, 450-13), and NT-3 (1 ng/mL, PeproTech, 450-03). OPCs were then used for IC 50 experiments. Pediatric cortical glioblastoma (SU-pcGBM) and diffuse intrinsic pontine glioma IV (SU-DIPG IV) maintained in our lab were acquired. The SU-pcGBM and SU-DIPG IV cells were cultured in a T75cm 2 flask (Nunc™ Cell Culture Treated 75 cm 2 EasYFlask™, Fisher Scientific). A defined, serum-free medium designated “Tumor Stem Media (TSM)” was used throughout, consisting of Neurobasal(-A), B27(-A), human-bFGF (20 ng/mL, Shenandoah Biotech,100-146), human-EGF (20 ng/mL, Shenandoah Biotech, 100-26), human PDGF-AA (10 ng/mL) and PDGF-BB (10 ng/mL) (Shenandoah, Biotech, 100-16 and 100-18) and heparin (2 ng/mL). When neurospheres and minimal space between growing cells were visible in the primary culture, the concentration of each cell line was determined using a hemocytometer (Hausser Scientific, Horsham, PA). NPCs were passaged every 4 days while SU-pcGBM and SU-DIPG IV cells were passaged every 2 weeks.

Wild-type CD57BL/6 mice (Charles River) were bred with CD1 mice (Charles River) for all experiments unless otherwise noted. All animals were housed in a 12-hour light:dark cycle with ad libitum access to food and water. Animals were housed 2-5 per cage. Both sexes were used equally for all studies. No animals were manipulated other than as reported for that experimental group, i.e., there was no history of drug exposures, surgeries or behavioral testing for the animals used other than that reported for the given experimental group. All procedures were performed in accordance with guidelines set in place by the Stanford University Institutional Care and Use Committee.

Human samples were collected as part of a routine autopsy protocol from a standardized location of frontal cortex/subcortical white matter in individuals treated with multi-agent chemotherapy during childhood/young adulthood and from age-matched, non-chemotherapy exposed control cases. No cases received cerebral radiotherapy. Samples were obtained in collaboration with the Department of Pathology at Stanford University (see Table S1 ). FFPE samples were stained immunohistochemically according to standard procedures on paraffin embedded sections (5 μm) (). A control stain of Nestin was used to ensure quality of tissue samples. The following antibodies were used: Olig-2 (1:100, Cell Marque, 387M-16) and Nestin (1:20000, Millipore, MAB5326). For each sample, 200X frames were captured using a Nikon Eclipse E1000 microscope with a SPOT Flex camera in both gray matter and white matter regions, for a total of 10 images/case. The total density of Olig2cells was determined by extrapolating number of Olig2cells by the total volume of tissue assessed. For fluorescent immunohistochemistry, frontal lobe samples from both cases were obtained as part of a rapid autopsy protocol for tumor tissue donation, fixed in 4% paraformadehyde and embedded in 30% sucrose. Tissue was then sectioned at 40 μm and processed for confocal microscopy as described below. Tissue was processed with goat anti-PDGFRα (1:250, R&D Systems, AF1062) and rabbit anti-NG2 (1:200, Sigma, HPA002951) and imaged at 400X using a Zeiss LSM 700. All human tissue studies were performed with informed consent and in accordance with Institutional Review Board (IRB)-approved protocols.

Method Details

Liquid chromatography tandem mass spectrometry (LC-MS/MS) Blood, kidney, liver, and frontal cortex were acquired 30 min post a single 100 mg/kg methotrexate injection from 5 mice. Mice were transcardially perfused with cold PBS followed by tissue sample dissection. Tissues samples were weighed and 1 volume of bullet blender beads (Next Advance) and 3 volume of Milli-Q water were added. Tissues were homogenized by a bullet blender (Next Advance) at 4°C according to manufacturer’s instruction. The neat stock solution of MTX was dissolved in DMSO at 5 mg/mL and further diluted in 50% methanol to prepare spiking solutions. To prepare spiked calibration curve, 25 μL of MTX spiking solutions (1-200 ng/mL for brain samples and 0.2-50 μg/ml for serum, kidney and liver samples) was mixed with 25 μL of blank tissue homogenate or serum. To prepare samples, the spiking solution was replaced by 25 μL of 50% methanol to make up the volume. After vortexing all standards and samples, 150 μL of methanol/acetonitrile 20:80 (v/v) containing the internal standard MTX-d3 was added to the mixture and vortexed vigorously for 1 min followed by centrifugation at 3,000 g for 10 min. The supernatant was diluted 3 times in Milli-Q water with 0.1% formic acid for brain samples and 50 times in 25% methanol with 0.1% formic acid for serum, kidney and liver samples. The LC-MS/MS system consisted of a QTRAP 4000 mass spectrometer (AB SCIEX) coupled to a Shimadzu UFLC system. LC separation was carried out on an Acclaim 120 C8 column (2.1 mm × 50 mm, 5 μm) (Dionex) at room temperature. The analysis time was 2.6 min. The injection volume was 10 μl. The flow rate was 0.3 mL/min. Mobile phase A consisted of 2 mM ammonium acetate and 0.2% formic acid in LCMS grade water. Mobile phase B consisted of 0.1% formic acid in LCMS grade acetonitrile. The HPLC elution program was as follows: 10% B (0.5 min)→40% B (linear increase in 1 min)→10% B (linear decrease in 0.1 min)→10% B (1 min). The mass spectrometer was operated in the positive mode with multiple-reaction monitoring (MRM) with the transition m/z 455.2→175.1 for MTX and m/z 458.2→311.1 for MTX-d3. Data acquisition and analysis were performed using the Analyst 1.6.1 software (AB SCIEX).

Chemotherapy treatment paradigm CD57BL/6:CD1 mice were given an i.p. injection of 100 mg/kg methotrexate (MTX) dissolved in PBS obtained from the pharmacy at Lucile Packard Children’s Hospital at Stanford University on P21, P28, and P35. For assessment of the effect of chemotherapy on proliferation, animals additionally received i.p. injections of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU; 40 mg/kg, Invitrogen, E10187) on P61, P62, and P63. Animals were then sacrificed (30 min post EdU injection) or assessed (see below) on P63 or P203.

Perfusion and immunohistochemistry Animals assessed using fluorescent immunohistochemistry were anesthetized with avertin and transcardially perfused with 10-15 mL PBS. Brains were post-fixed in 4% paraformaldehyde (PFA) overnight at 4°C prior to cryoprotection in 30% sucrose. Brains were embedded in O.C.T. (Tissue-Tek) and sectioned at 40 μm in the coronal plane using a sliding microtome (Leica, HM450). For immunohistochemistry involving EdU, sections were stained using the Click-iT EdU cell proliferation kit (Thermo Fisher, C10424) and protocol to expose EdU labeling prior to blocking. For all other stains, sections were incubated directly in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) for 1 hr at room temperature and then incubated in primary antibodies overnight. Goat anti-PDGFRα (1:500; R&D Systems AF1062), rabbit anti-Iba1 (1:2000; Wako 019-19741), rat anti-CD68 (1:200; Abcam AB53444), rabbit anti-Olig1 and mouse anti-Olig1 (1:5000; 1:500, respectively, a generous gift from Dr. John Alberta), mouse anti-CC1 (1:20 and incubated for 7-10 days; Millipore OP-80), rabbit anti-cleaved caspase-3 (1:500; Cell Signaling 9661S) and rat anti-MBP (1:200; Abcam AB7349) were diluted in 1% solution (1% normal donkey serum, 0.3% Triton X-100 in TBS) and incubated overnight at 4°C. All antibodies have been validated in the literature and/or in Antibodypedia for use in mouse immunohistochemistry. To further validate the antibodies in our hands, we confirmed that each antibody stained in the expected cellular patterns and brain-wide distributions. Secondary-only stains were performed as negative controls. The following day, sections were rinsed 3 times in 1X TBS and incubated in secondary antibody solution (1:500) and DAPI (1:1000) in 1% solution at 4°C overnight. The following secondary antibodies were used, Alexa 488 donkey anti-goat (Jackson ImmunoResearch), Alexa 594 donkey anti-goat (Jackson ImmunoResearch), Alexa 647 donkey anti-mouse (Invitrogen) or Alexa 647 donkey anti-rabbit (Jackson ImmunoResearch). The next day, sections were rinsed 3 times in TBS and mounted with Prolong Gold plus DAPI mounting medium (Invitrogen).

RNAScope Animals were sacrificed at P62-64 and perfused with HBSS. Brains were removed and immediately placed in OCT and frozen in liquid nitrogen. Tissue was stored at −80°C until sectioned on a cryostat at a thickness of 16 μm. Before performing RNAScope, slides were transferred directly from −80°C to 4% PFA/HBSS on ice for 15 min. Sections were then successively dehydrated in 50% ethanol, 70% ethanol, and twice in 100% ethanol for 5 min each and allowed to dry before treatment for 5 min with Protease IV from the RNAScope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics, 320850). Slides were washed twice with HBSS before proceeding to probe hybridization. RNAScope was performed according to Fluorescent Multiplex Reagent Kit protocol using RNAScope probes against Slc1a3 (Glast; Advanced Cell Diagnostics, 430781) and Cxcl10 (Advanced Cell Diagnostics, 408921-C2). Slides were mounted in Prolong Gold and imaged on either a Zeiss LSM700 or LSM800 at 400X magnification within 72 hr of hybridization. Z stacks were acquired for three counting frames throughout the corpus callosum of the frontal cortex and a maximum intensity image was generated for each image. The number of Cxcl10 puncta per Glast+ cell was quantified by a blind rater for each image. Those Glast+ astrocytes with more than 8 Cxcl10 puncta/cell were considered pan-reactive.

Confocal imaging For fluorescent immunohistochemistry, two representative sections, one anterior and one posterior to the formation of the genu of the corpus callosum were selected for each animal. Z stacks were acquired using a Zeiss LSM 700 or LSM 800 for five counting frames taken at 200X (320 μm x 320 μm frame) between the two sections and a maximum intensity image was generated for each image. For each section, a superficial frame was captured to include layers I and II of the premotor cortex (M2) and a deep cortical frame was captured to include layers V and VI of the premotor cortex. For the white matter quantification, each section was imaged at the cingulum of the corpus callosum and half-way to midline within the corpus callosum. The posterior section included a frame taken at the genu of the corpus callosum.

Volume measures Estimated volume measurements for the corpus callosum were obtained using the Cavalieri Estimator function on a MBF Bioscience StereoInvestigator version 11.01.2. A 1:6 series of coronal brain sections were stained with MBP antibody (myelin basic protein). Using the 10X objective, the Cavalieri Estimator probe was used to trace the corpus callosum; the boundaries were determined using published mouse anatomical guides. A total of 13 sections were analyzed per treatment group.

Electron microscopy Four weeks (P63) or 6 months (P203) after the cessation of treatment, mice were sacrificed by transcardial perfusion with Karnovsky’s fixative: 2% glutaraldehyde (EMS 16000) and 4% paraformaldehyde (EMS 15700) in 0.1M sodium cacodylate (EMS 12300), pH 7.4. Region containing premotor cortex (M2) and projections to the corpus callosum was resected from the brain and post-fixed in Karnovsky’s fixative for at least 2 weeks. Transmission electron microscopy was performed in the region of the M2 subcortical fibers as they leave cortical layer VI and enter the corpus callosum. The samples were then post-fixed in 1% osmium tetroxide (EMS 19100) for 1 hr at room temperature, washed 3 times with ultrafiltered water, then en bloc stained for 2 hr at room temperature. Samples were then dehydrated in graded ethanol (50%, 75%, and 95%) for 15 min each at 4°C; the samples were then allowed to equilibrate to room temperature and were rinsed in 100% ethanol 2 times, followed by acetonitrile for 15 min. Samples were infiltrated with EMbed-812 resin (EMS 14120) mixed 1:1 with acetonitrile for 2 hr followed by 2:1 EMbed-812:acetonitrile for 2 hr. The samples were then placed into EMbed-812 for 2 hr, then placed into TAAB capsules filled with fresh resin, which were then placed into a 65°C oven overnight. Sections were taken between 75 and 90 nm on a Leica Ultracut S (Leica, Wetzlar, Germany) and mounted on Formvar/carbon coated slot grids (EMS FCF2010-Cu) or 100 mesh Cu grids (EMS FCF100-Cu). Grids were contrast stained for 30 s in 3.5% uranyl acetate in 50% acetone followed by staining in 0.2% lead citrate for 30 s. Samples were imaged using a JEOL JEM-1400 TEM at 120kV and images were collected using a Gatan Orius digital camera. With experimenters blinded to sample identity and condition, axons were analyzed for g-ratios calculated by dividing the axonal diameter by the corresponding axonal-plus-sheath diameter (diameter of axon/diameter of axon + myelin sheath) at 4000X using ImageJ software. For each animal, approximately 100 axons were scored. Statistics for g-ratios were calculated on a per animal basis.

IC 50 Testing 96 well plates (Corning Life Sciences, Corning, NY) were coated with 50 μL of PLL (Poly-L-Lysine, 10 μg/mL, Sigma-Aldrich, St. Louis, MO) for 30 min, and then rinsed 3 times with PBS, each rinse at least 5 min in duration. After the last rinse with PBS, the plates were stored in the incubator at 37°C for at least 3 hr. Cells were plated at concentration of 5000 cells/well in 100 μL of their respective medium. Triplicates for each concentration of MTX (diluted in cell medium appropriate for each cell line) from 10 mM of MTX to 10 pM (decreasing in concentration by an order of magnitude for every triplicate), were tested to best determine the IC 50 for each cell line. The cells were then incubated for 24 or 48 hr at 37°C. CellTiter-Glo reagent (Promega, G9243) was added at a 1:1 ratio (100 μL) and allowed to incubate for 10 min at room temperature before measuring luminescence using a plate reader (Thermoscientic Varioskan LUX multimode microplate reader). Average luminescence for each cell line at every concentration of MTX tested was normalized to the controls (no MTX exposure (positive control) and no cells plated (negative control)). Using this procedure, it was possible to determine the extent of cell death for every concentration of MTX tested and extrapolate the concentration of MTX exposure leading to 50% cell death of each cell line.

Syngeneic Transplantation of OPCs eGFP (C57BL/6-Tg(CAG-EGFP)1Osb/J) mice (The Jackson Laboratory) on a CD57/BL6 background were bred with CD1 mice to produce animals that were syngeneic with the juvenile MTX or PBS treated mice used in this study. P6-8 GFP+ mice were used to sort PDGFRα+ cells by FACS from the frontal cortex (see above). Sorted GFP+/PDGFRα+ cells were immediately stereotactically transplanted unilaterally into deep cortical M2/cingulum of the corpus callosum (A-P: +0.7 mm; M-L: 0.9 mm; D-V: −2.0 mm) of previously treated MTX or PBS animals at P63 (four weeks post-treatment). Cells were injected at a concentration of 20,000 cells/μl. Cells were allowed to engraft for 10 days before animals were transcardially perfused (see above).

Behavioral analysis CatWalk To investigate the effect of juvenile chemotherapy exposure on motor output, the CatWalk gait analysis system (Noldus, Netherlands) was used. To ensure consistent running, mice were acclimated to handling for several weeks before recording, and all tests were run in a dark room. Animals were tested four weeks (P63) after the cessation of the chemotherapy treatment paradigm previously described. No behavioral training on the CatWalk apparatus was performed. Behavioral testing was performed during the light cycle in a dark room with red light. Four successful runs (with success being characterized by variation under 60%, lasting no more than 5 s, and consistent movement) were processed with the CatWalk XT 9.0 software. Our previous work suggests that swing speed, as a sensitive measure of motor system function, is selectively altered if myelination is affected within the premotor circuit. We did not expect to see a difference in stride length. In this study, only the following parameters were analyzed: Swing speed: the speed (cm/s) of the paw during limb swinging. Swing speed was calculated as the average of the left and right forepaw swing speeds. Stride length: the distance (cm) between successive placements of the same paw. Calculation of stride length is based on the X-coordinates of the center of the paw print of two consecutive placements of the same paw during maximum contact of the paw with the glass floor. Open Field test Social anxiety was analyzed using a modified version of the open field test that focused on an animal’s willingness to explore objects placed in the center of an open field. All mice were exposed to the same MTX/PBS paradigm at P21-35 as described above. Animals were handled at P62 for 10 min. After handling, the mice were placed in the experimental chamber to acclimate for 20 min. The experimental chamber was 61cm x 61cm x 61cm made of opaque Plexiglas. A camera was mounted 115 cm above the chamber to record the animal’s behavior. The test was conducted during the animal’s light phase, in a dark room with only red light. On the day of testing (P63) the mice were handled for two min, and then placed in the experimental chamber with two identical Lego objects (of approx. 5 cm in height). The mouse was allowed to explore the arena for 5 min. After the testing was completed the mice were weighed, as a measure of health, to allow the groups to be compared. The camera footage was then analyzed (using CowLog analysis software). The animal’s willingness to explore the center of the arena with the objects was assessed. Novel Object Recognition Test Cognition was analyzed using a modified version of the novel object recognition task (NORT) that focused on the attentional component of the task. The test was modified so that the duration between the training and testing phase was shortened, this was to ensure that the test placed a greater cognitive load on short-term memory, attention and frontal lobe function rather than long-term memory and hippocampal function. All mice were exposed to the same MTX/PBS paradigm at P21-35 as described above. Animals were handled daily for the week leading up to the test for 2 min. After handling, the mice were then placed in the experimental chamber on P62 or P202 to acclimatize for 20 min prior to testing on P63 or P203, respectively. The set up consisted of an opaque Plexiglas experimental chamber 61cm x 61cm x 61cm, and a camera mounted 115 cm above the chamber. The test was conducted during the animal’s light phase, in a dark room illuminated with only red light. On the day of testing (P63 or P203) the mice were handled for 2 min and then placed in the chamber to acclimate for 20 min before being returned to the home cage for another 5 min. Mice were then placed in the experimental chamber with two identical Lego objects (of approx. 5 cm). Each time the mouse was placed into the chamber, the mouse was facing the opaque wall, with the animal’s tail in the direction of the objects. During the training phase, the mouse was allowed to explore the identical objects for 5 min. The mouse was once again returned to the home cage for 5 min and the experimental chamber and the objects were cleaned with 70% ethanol. During this time, one cleaned object from the sample phase was placed back into the experimental chamber along with a new Lego object (of approx. 5 cm) for the novel object phase. During the testing phase, the mouse was returned to the experimental chamber and allowed to explore for 10 min. Leger et al., 2013 Leger M.

Quiedeville A.

Bouet V.

Haelewyn B.

Boulouard M.

Schumann-Bard P.

Freret T. Object recognition test in mice. The objects used as novel and familiar were counterbalanced, as was the position of the novel object from trial to trial, animal to animal. All of the Lego objects used in the behavioral paradigm were piloted to ensure that there was no bias, or object preference for the animals. The camera footage was then analyzed (using CowLog analysis software), and any exploratory head gestured within 2 cm of the Lego object, including sniffing and biting were considered object investigation, but not sitting on the object, or casual touching of the object in passing (). Only animals that explored the objects for a minimum of 20 s for P63 or 10 s for P203 were included in the analysis. After the testing was completed the mice were weighed, as a measure of health, to allow the groups to be compared. The Recognition Ratio was determined by taking the ratio of the amount of time spent investigating one object compared to the total time spent investigating both objects (i.e., time spent with Novel Object / (time spent with Novel Object + time spent with Familiar Object)).

Microfluidic qRT-PCR Liddelow et al., 2017 Liddelow S.A.

Guttenplan K.A.

Clarke L.E.

Bennett F.C.

Bohlen C.J.

Schirmer L.

Bennett M.L.

Münch A.E.

Chung W.S.

Peterson T.C.

et al. Neurotoxic reactive astrocytes are induced by activated microglia. Total RNA was extracted from immunopanned cells using the RNeasy Plus kit (QIAGEN) and cDNA synthesis performed using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) according to supplier protocols. We designed primers using NCBI primer blast software ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and selected primer pairs with least probability of amplifying nonspecific products as predicted by NCBI primer blast. We tested the specificity of the primer pairs by PCR with rat and mouse whole-brain cDNA (prepared fresh), and examined PCR products by agarose gel electrophoresis. For microfluidic qRT-PCR, 1.25 μL of each cDNA sample was pre-amplified using 2.5 μL of 2x Taqman pre-amplification master mix (Applied Biosystems) and 1.25 μL of the primer pool (0.2 pmol each primer/μL). Pre-amplification was performed using a 10 min 95°C denaturation step and 14 cycles of 15 sec at 95°C and 4 min at 60°C. Reaction products were diluted 1:5 in TE Buffer (Teknova). Five microliters from a sample mix containing pre-amplified cDNA and amplification Master mix (20 mm MgCl2, 10 mm dNTPs, FastStart Taq polymerase, DNA binding dye loading reagent, 50 × ROX, 20 × Evagreen) was loaded into each sample inlet of a 96.96 Dynamic Array chip (Fluidigm) and 5 μL from an assay mix containing DNA assay loading reagent, as well as forward and reverse primers (10 pmol/ μL) was loaded into each detector inlet. The chip was then placed in the NanoFlexTM 4-IFC Controller (Fluidigm) for loading and mixing. After loading, the chip was processed in the BioMarkTM Real-Time PCR System (Fluidigm) using a cycling program of 10 min at 95°C followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec and 72°C for 30 sec. After completion of qPCR, a melting curve of amplified products was determined. Data were collected using BioMarkTM Data Collection Software 2.1.1 build 20090519.0926 (Fluidigm) as the cycle of quantification (Cq), where the fluorescence signal of amplified DNA intersected with background noise. Data were corrected for differences in input RNA using the geometric mean of the reference gene Rplp0. Data preprocessing and analysis was completed using Fluidigm Melting Curve Analysis Software 1.1.0 build 20100514.1234 (Fluidigm) and Real-time PCR Analysis Software 2.1.1 build 20090521.1135 (Fluidigm) to determine valid PCR reactions. Invalid reactions were removed from later analysis. All primer sequences for rat and mouse were used previously (; see Table S2 ).

Standard qRT-PCR Liddelow et al., 2017 Liddelow S.A.

Guttenplan K.A.

Clarke L.E.

Bennett F.C.

Bohlen C.J.

Schirmer L.

Bennett M.L.

Münch A.E.

Chung W.S.

Peterson T.C.

et al. Neurotoxic reactive astrocytes are induced by activated microglia. Total RNA was extracted and cDNA synthesized as above. Quantitative RT-PCR was run using 2 μL cDNA and SYBR green chemistry (Applied Biosystems/ThermoFisher Scientific) using supplier protocol and a cycling program of 2 min at 95°C followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec on a Mastercycler epgradient S (Eppendorf). After completion of qPCR, a melting curve of amplified products was determined. Data were collected using a Mastercycler ep realplex v2.2 (Eppendorf). All primer sequences for rat and mouse were used previously (; see Table S2 ).