Determination of Col1A2 gene transcription Sample Clauses

Determination of Col1A2 gene transcription. The COL1A2-luciferase construct contains sequences from -353 to +58 bp of the human COL1A2 promoter fused to the luciferase reporter gene. NIH3T3 fibroblast cells will be cultivated at a density of 5 x 104 cells/ml for 24 h at 37ºC and transfected with the COL1A2-luc plasmid using Roti-Fect (Xxxx Xxxx) following the manufacturer´s recommendations. Twenty-four h later the cells will be treated with the test compounds at several concentrations and TGFβ1 (10 ng/ml) as positive control for 12h. The cells will be will behed with PBS and lysed in 100µl lysis buffer containing 25 mM Trisphosphate (pH 7.8), 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min. Luciferase activity and protein concentration will be measured in the cell lysates to calculate the RLU/mg of protein and the results expressed as the percentage of induction considering the value obtained with TGFβ1 as 100% activation.
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