Human Microglia Cell Culture
The immortalized human microglia-SV40 cell line derived from primary human microglia was purchased from Applied Biological Materials Inc. (ABM Inc.; Richmond, BC, Canada) and cultured in Prigrow III medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in type I collagen-coated T25-flasks (ABM Inc.). Microglia-SV40 maintain their phenotype and proliferation rates for over 10 passages, during which all experiments were performed using multiple microglia thaws and sub-cultured cells. Experiments were carried out in type I collagen-coated plates (BD PureCoat™ ECM Mimetic Cultureware Collagen I peptide plates, Becton Dickinson, Bedford, MA). Cell viability was determined by trypan blue (0.4%) exclusion.
Microglia Treatments
Human microglia were stimulated with 1–10 ng/mL of recombinant full-length SARS-CoV-2 S (Abcam, Waltham, MA and/or GeneTex, Irvine, CA), 1–10 ng/mL of RBD (Abcam, Waltham, MA and/or GeneTex, Irvine, CA), and/or pretreated with 2 µg/mL of anti-TLR2 Ab (InvivoGen, San Diego, CA), 2 µg/mL of anti-TLR4 Ab (InvivoGen, San Diego, CA) or 2 µg/mL of anti-ACE2 Ab (InvivoGen, San Diego, CA). LPS (10 ng/mL) and NT (10 nM) were used as positive controls.
Proinflammatory Mediator Release
Microglia (0.5 × 105 cells/ well) were seeded in 12-well, type I collagen or poly-L-lysine–coated plates (Becton Dickinson, Bedford, MA) for 24 h before stimulation with full-length SARS-CoV-2 S (1–10 ng/mL) or RBD (1–10 ng/mL) was carried out. For selected experiments, microglia were pretreated with anti-TLR2 Ab or anti-TLR4 Ab or anti-ACE2 Ab for 24 h before stimulation with full-length SARS-CoV-2 S or RBD. After 24 h, supernatant fluids were collected and concentrations of IL-1β, CXCL8, IL-6, TNF-α, MMP9, S100B and IL-18 were measured using commercially available ELISA DuoSet kits (DY201, DY208, DY206-05, DY210-05, DY911-05, DY1820-05 and DY318-05 respectively) from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. Control cells were treated with equal volume of culture medium in all experiments. The detection limits of IL-1β were 3.91–250 pg/mL, CXCL8 were 31.3–2000 pg/mL, IL-6 were 9.38–600 pg/mL, TNF-α were 15.6–1000 pg/mL, MMP9 were 31.3–2000 pg/mL, S100B were 46.9–3000 pg/mL and IL-18 were 11.7–750 pg/mL.
Statistical Analysis
All in vitro conditions were performed in triplicate, and all experiments were repeated at least three times (n = 3). Results are presented as mean ± standard error of the mean (SEM). Differences between two groups were assessed using the Student’s t-test. Comparisons among at least three groups were tested by one-way analysis of variance (ANOVA), and then post hoc comparisons to determine significant differences between several experimental groups and the control group and between two groups were performed using Dunnett's test and Bonferroni test, respectively. Differences with p-values less than 0.05 were considered statistically significant. All analyses were performed using Graph Pad Prism 5.