Primary human microglia isolated from cortex (Celprogen, #37089-01) were cultured on Matrigel-coated 12-well plates at 5 × 105 cells per well in iMGL differentiation media at 37 °C in 5% CO2. Cells (up to passage 5) were transfected for 48 h with dox-inducible GFP-tagged Gateway-generated Piggybac expression constructs with inserted cDNA encoding human HIF1A and/or ELF3 open reading frames using Glial Mag transfection kit (Oz Biosciences, #GL00500) according to manufacturer’s instructions. Primary microglia were co-transfected with plasmids encoding Hybase transposase and rtta at a ratio of 1:2:2 (Hybase: rtta: ORF).
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