Microglia Proliferation Quantification

Proliferation was quantified using the CyQUANT™ NF assay (Thermo Fisher Scientific; Cat # C35006), as before (Lam and Schlichter, 2015 (link)). Microglia were seeded at 2–3 × 10⁴ cells/well of a 96-well flat-bottom plate in MEM with 2% FBS and allowed to settle for 24 h, then left untreated (control) or stimulated with TGFβ1 for 24 h. The detection dye was added to each well for 30 min (37°C, 5% CO₂), and the fluorescence intensity was measured using a multi-label plate reader (Victor³ 1420, Perkin Elmer, Woodbridge, ON, Canada), with excitation at 485 nm and emission at 535 nm. Duplicate readings were taken for 0.1 s at 3 mm from the bottom of the plate, averaged, and then the background was subtracted before normalizing to the control group.