The transfection mix was removed after 4 8 h, and cells were washed with PBS and cultured in supplemented DMEM.
After 4 h incubation, the transfection mix was removed and the cells were washed with fresh medium 4 times.
Following 24 h of transfection, the DNA-Lipofectamine mix was removed and the cultures were supplied with fresh medium containing either 1-NMPP1 or the vehicle DMSO for another 24 h.
After incubation, the acidic acid/glycerol mix was removed completely, and 150 µl of mounting medium (Hoyer's medium) was added to the larvae where they were left to soak for 15 30 mins.
Excess reaction mix was removed by sonication in chloroform and IPA for 5 min each.
After 3 hours incubation, transfection mix was removed and fresh supplemented DMEM was added.
Immediately, at time zero, then at 3, 6, 9, 15 and 30 min, an aliquot (50 μL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to stop the reaction.
Immediately, at t0, then at 3, 6, 9, 15, and 30 min an aliquot (50 μL) of the incubation mixture was removed and mixed with MeCN (100 μL) to stop the reaction.
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Immediately at time zero, then at 3, 6, 9, 15, and 30 min, aliquots (50 μL) of the incubation mixture were removed and mixed with MeCN (100 μL) to stop the reaction.
After 3 days, the medium was replaced with the differentiation medium without FGF1 and heparin, and after 6 days Rosiglitazone/BRL49653, MIX, and DEX was removed from the medium.
