Fig 2: Assay measurements are slightly higher in serum than in plasma and are reduced in the presence of hemolysis. Blood was collected from mice 6 h after isoflurane exposure (sham) (i) or isoflurane exposure and TBI (ii) by cardiac puncture into tubes with (plasma) and without (serum) EDTA. a Plasma and serum were isolated by centrifugation and were assayed in duplicate. Differences in concentrations by matrix were analyzed by paired t test, ****p < 0.0001. b Plasma specimens were spiked with 0%, 5%, 25%, or 50% red blood cells, frozen at - 80 °C for 1 h, thawed, and assayed in duplicate. The effect of hemolysis on measured concentrations was analyzed by one-way ANOVA with repeated measures (exact p values are shown below graphs) followed by Dunnett’s multiple comparisons test, no significant differences from neat plasma were found. Mean plasma GFAP concentrations for each mouse are plotted with points representing mean and error bars ±SD of duplicates, in some cases error bars are smaller than the symbol. TBI: traumatic brain injury, EDTA: ethylenediaminetetraacetic acid, SD: standard deviation

Fig 3: Plasma GFAP levels are elevated in aged APP/PS1 mice and APP/PS1 mice lacking apoA-I. a Female APP/PS1 mice (on a mixed C3H/Bl6 background) were aged to 3, 6, 9, 12, 18, or 24 months old then plasma specimens were collected by cardiac puncture. Mean plasma GFAP concentrations of each group are plotted with points representing individual mouse plasma samples and error bars representing ±SD. The effects of age and APP/PS1 genotype on plasma GFAP concentration were analyzed by two-way ANOVA (exact p values below graph) followed by Tukey’s multiple comparisons test across genotypes (exact p values below graph) or Sidak’s multiple comparison’s test within each genotype (detailed within graph, **p < 0.01, ***p < 0.001). b Male and female apoA-I hemizygous (apoA-IHEM) or knockout (apoA-IKO) mice with or without APP/PS1 transgenes (on a C57Bl/6 background) were aged to 12 months old then plasma specimens were collected by cardiac puncture. Mean plasma GFAP concentrations of each group are plotted with points representing individual mouse plasma samples and error bars representing ±SD. The effects APP/PS1 genotype and apoA-I genotype on plasma GFAP concentration were analyzed by two-way ANOVA (exact p values below graph) followed by Sidak’s multiple comparisons test (detailed within graph *p < 0.05, **p < 0.01). apoA-I: apolipoprotein A-I, SD: standard deviation

Fig 4: The assay has a wide quantitative dynamic range from 25 pg/mL to at least 16,533 pg/mL and a lower limit of detection of 9 pg/mL. Eight plasma samples with a very low or b very high expected GFAP concentrations were assayed in duplicate and c CV of duplicates were determined. d 16 blank replicates (1% BSA in PBS) were assayed to calculate an estimated lower limit of quantification and lower limit of detection as described in Table 6. a,b,d Mean concentration of replicates is plotted with each point representing an individual sample replicate and error bars representing ± SD. c Points represent mean sample concentration and CV of duplicates. a–c Circles represent samples from female mice, squares represent samples from male mice. AU: arbitrary units, CV: coefficient of variation, SD: standard deviation, BSA: bovine serum albumin, PBS: phosphate-buffered saline, ECL: electrochemiluminescence

Fig 5: The assay shows high dilution linearity over a wide range of calibrator and plasma specimen dilutions. Assay calibrator was spiked into 3 individual plasma samples with very low endogenous GFAP to a concentration of 2,000,000 pg/mL. Serial dilutions from 10-fold to 1,000,000-fold were made and samples at each dilution step were assayed in duplicate. a Percent recovery of samples spiked with calibrator plotted against dilution factor. b ECL signal of samples spiked with calibrator were plotted against the dilution factor. Serial dilutions were performed from 2-fold to 64-fold on 3 plasma samples with high endogenous GFAP concentrations. c Percent recovery of diluted plasma samples plotted against dilution factor. d ECL signal of diluted plasma samples and calibrator plotted against dilution factor. Points represent mean concentration of duplicates and error bars represent ±SD. For some points, the error bars are shorter than the height of the symbol and therefore are not shown. AU: arbitrary units, ECL: electrochemiluminescence