Human primary microglia culture and transfection

AG Alexandra Grubman
XC Xin Yi Choo
GC Gabriel Chew
JO John F. Ouyang
GS Guizhi Sun
NC Nathan P. Croft
FR Fernando J. Rossello
RS Rebecca Simmons
SB Sam Buckberry
DL Dulce Vargas Landin
JP Jahnvi Pflueger
TV Teresa H. Vandekolk
ZA Zehra Abay
YZ Yichen Zhou
XL Xiaodong Liu
JC Joseph Chen
ML Michael Larcombe
JH John M. Haynes
CM Catriona McLean
SW Sarah Williams
SC Siew Yeen Chai
TW Trevor Wilson
RL Ryan Lister
CP Colin W. Pouton
AP Anthony W. Purcell
OR Owen J. L. Rackham
EP Enrico Petretto
JP Jose M. Polo
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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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