MH100-Tk-luciferase reporter vector was co-transfected into HEK293 as well as C2C12 cells along with Renilla luciferase and a vector expressing fusion protein of yeast-derived Gal4 DBD and either full-length or mutant NFIA TAD. Cells were harvested 24 hours after transfection and dual luciferase reporter was assayed by TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold). Reporter luciferase activity was normalized to the Renilla luciferase activity.
Tristar2 lb 942 modular multimode microplate reader
Manufactured by Berthold Technologies
Sourced in Germany
The TriStar2 LB 942 Modular Multimode Microplate Reader is a laboratory instrument designed for the detection and quantification of various biomolecules and compounds in microplate formats. It offers flexible detection modes, including absorbance, fluorescence, and luminescence measurements.
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5 protocols using tristar2 lb 942 modular multimode microplate reader
1
Gal4-NFIA Transcription Assay
2
Caspase-3/7 Activity Measurement
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Caspase-3/7 activities were measured using Caspase-Glo® 3/7 Assay Systems (Promega GmbH) according to the manufacturer’s instructions. Cells were seeded in 96-well plates and were infected the next day when cells reached a confluence of about 80% at MOI of 0.1 and 0.01. Twenty-four h after infection luminescence was measured using the TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany).
3
Cell Viability Measurement by XTT Assay
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Cell viability was assessed by using Cell Proliferation Kit (XTT) (Promega GmbH, Walldorf, Germany) according to the manufacturer’s instructions. Cells were seeded in 96-well plates and infected the next day when they reached a confluence of about 80%. Twenty-four h, 48–72 h after infection absorbance levels were measured using the TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). As negative control, cells were treated with 50 µl 5% Triton X-100 solution.
4
Regulation of CCL2 Promoter Activity
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The pGL4.19-mCcl2 promoter luciferase reporter vector was cotransfected into WT or NFIA-Tg adipocytes with the Renilla luciferase reporter vector using lipofectamine 2000. When indicated, pcDNA 3.1 NFIA or pCXN2 FLAG-NF-κB p65 was also transfected into cells. Cells were harvested 24 h after transfection, and dual luciferase reporter activity was assayed using the TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold). Reporter luciferase activity was normalized to the Renilla luciferase activity. The pGL4.19 mCcl2 promoter luciferase reporter vector was a gift from Masashi Kuroda and Hiroshi Sakaue (Tokushima University, Tokushima, Japan), and pCXN2 FLAG-NF-κB p65 expression vector was a gift from Hideyuki Yanai (The University of Tokyo, Tokyo, Japan).
5
Stable 3T3-L1 PPARγ and STAT3 Reporter Cells
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Stable 3T3-L1 PPARγ and STAT3 reporter cell lines were generated as follows. Briefly, consensus binding sites of PPARγ and STAT3 (AGGACAAAGGTCA for PPARγ and TTTCCGGGAA for STAT3) were identified using the TRANSFAC public database. The response element (RE) oligonucleotides, containing three consensus binding sequences, were cloned into the GLuc-DRE2-viral vector, wherein GLuc is regulated by a minimal promoter [14 (link)]. The expression of GLuc is induced when a target transcription factor binds to its consensus binding site. Thereafter, stable 3T3-L1 PPARγ and STAT3 reporter cell lines were generated through lentiviral transduction.
To profile the activation of PPARγ and STAT3, the reporter cells were differentiated in 12-well plates as described above. The supernatant was harvested on day 1, day 3, and day 5 post-differentiation induction and used to measure GLuc activity. GLuc activity (Relative Light Units; RLU) was assessed using the Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold technologies, Bad Wildbad, Germany) in accordance with the manufacturers’ instructions. After normalization with cell density, relative fold changes were determined at each time point.
To profile the activation of PPARγ and STAT3, the reporter cells were differentiated in 12-well plates as described above. The supernatant was harvested on day 1, day 3, and day 5 post-differentiation induction and used to measure GLuc activity. GLuc activity (Relative Light Units; RLU) was assessed using the Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold technologies, Bad Wildbad, Germany) in accordance with the manufacturers’ instructions. After normalization with cell density, relative fold changes were determined at each time point.
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