Microglial Viability and Proliferation Assays

Microglia (6 × 10⁴ cells/coverslip) were unstimulated or stimulated for 24 h with a cytokine: IL-4 or IL-10. When a Kir2.1 inhibitor (Ba²⁺ or ML133) was used, it was added at the same time as the cytokine. To examine viability, microglia were incubated with propidium iodide (500 nM, Invitrogen) for 1 h (37°C, 5% CO₂) before fixing with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 10 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 for 5 min, and washed with PBS (3×, 5 min each), and stained with FITC-conjugated tomato lectin (TL; 1:500, 15 min), and the nuclear dye, 4′, 6-diamidino-2-phenylindole (DAPI; 1:3000, 5 min; Invitrogen). After washing (3×, 5 min each), coverslips were mounted on glass slides using Dako mounting medium (Dako, Glostrup, Denmark). Five random fields were imaged at 20× or 40× magnification using the deconvolution microscope (DECON; Carl Zeiss, Jena, Germany). Counts of dead microglia (cells double-labeled with PI and DAPI) were normalized to the total number of DAPI-positive cells in 5 fields of view for each treatment condition.
For proliferation, we used the CyQUANT NF assay (Invitrogen). Microglia were seeded at 4 × 10⁴ cells/well of a 96-well flat-bottom plate and cultured in MEM with 2% FBS for 1–2 days (37°C, 5% CO₂). Then, they were stimulated for 24 h with IL-4 or IL-10, with or without a Kir2.1 channel inhibitor (Ba²⁺ or ML133). The dye-binding solution was added to the wells, incubated for 30 min (37°C, 5% CO₂), and then the fluorescence intensity was measured using a multi-label plate counter (Victor³ 1420, Perkin Elmer, Woodbridge, ON, Canada), with excitation at 485 nm and emission at 535 nM. Readings were taken for 0.1 s at 3 mm from the bottom of the plate, in triplicate and averaged. For analysis, the readings with each Kir2.1 blocker were normalized to the untreated unstimulated (control) group.