Mir 205 mimic

Astrocytes were cultured in either normal glucose (5 mM) or high glucose (25 mM) DMEM depending upon the experiment. Before transfection, 100,000 to 200,000 astrocytes were seeded in medium size petri dishes (Falcon cat. no. 35046) in 1.5ml of DMEM containing FBS and antibiotics and the appropriate concentration of glucose. Cells were transfected using HiPerfect (Qiagen, Cat. No. 301705) and the fast-forward protocol for transfection of adherent cells recommended by the manufacturer. Astrocytes grown in high glucose DMEM were treated with the miRNA205 inhibitor (Qigaen Cat. No. MIN0000878) or HiPerfect alone (mock-transfected), whereas astrocytes grown in normal glucose DMEM were treated with the miR-205 mimic (Qiagen, Cat. No. MSY0000878) or HiPerfect alone (mock-transfected). Briefly, 10 μl of 20 μM miR-205 inhibitor and 12μL of HiPerfect or 10 μl of 20 μM miR-205 mimic and 24μl of HiPerfect were diluted in 400μL of culture medium without serum. The mix was incubated for 10 minutes at room temperature to allow the formation of transfection complexes. The complexes were then added to the cells in a drop-wise fashion giving a final volume of 2mL and a final concentration of 100nM for the mimic and inhibitor. The plate was gently swirled to evenly distribute the transfection complexes and cells were incubated at 37°C for three days.